Hindawi Publishing Corporation Psyche Volume 2012, Article ID 470436, 16 pages doi:10.1155/2012/470436
Review Article An Insight into the Sialomes of Bloodsucking Heteroptera
JoseM.C.Ribeiro,TeresaC.Assumpc´ ¸ao,andIvoM.B.Francischetti˜
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
Correspondence should be addressed to JoseM.C.Ribeiro,´ [email protected]
Received 27 January 2012; Accepted 17 April 2012
Academic Editor: Mark M. Feldlaufer
Copyright © 2012 Jose´ M. C. Ribeiro et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Saliva of bloodsucking arthropods contains dozens or hundreds of proteins that affect their hosts’ mechanisms against blood loss (hemostasis) and inflammation. Because acquisition of the hematophagous habit evolved independently in several arthropod orders and at least twice within the true bugs, there is a convergent evolutionary scenario that creates a different salivary potion for each organism evolving independently to hematophagy. Additionally, the immune pressure posed by their hosts creates additional evolutionary pressure on the genes coding for salivary proteins, including gene obsolescence, which opens the niche for coopting new genes (exaptation). In the past 10 years, several salivary transcriptomes from bloodsucking Heteroptera and one from a seed- feeding Pentatomorpha were produced, allowing insight into the salivary potion of these organisms and the evolutionary pathway to the blood-feeding mode.
1. Introduction liquefying insoluble or viscous tissues or by helping to seal the feeding site in sap suckers, were the phloem is under The order Hemiptera (bugs) comprises hemimetabolous very high pressure [34]. Saliva has a biochemical role in insects having in common tubular mouthparts specialized aiding digestion of the meal, just as we have amylase in our for sucking liquid diets. The diet of Hemiptera is varied, own saliva; most remarkably, predacious bugs inject a highly the majority feeding on plants by either tapping the vessels hydrolytic cocktail into their victims that is digested while conducting sap or by lacerating and flushing tissues such as the prey is held by the predator, which can then later suck leaves or seeds. Within the suborder Heteroptera (true bugs), the liquefied victim and discard it as an empty shell. Saliva predatory feeding (with killing of the victim) also occurs, can also work pharmacologically by preventing the hosts’ mostly targeting other insects but also including small defense mechanisms against tissue loss, as occurs with the vertebratessuchasgiantwaterbugsandtoadbugs,aswell saliva of blood-feeding insects in preventing blood clotting, as blood or hemolymph feeding (without killing the victim) for example [35]. from vertebrate and invertebrate animals. The mouthparts Among the Heteroptera, the blood-feeding habit evolved are not only important for channeling the liquid meal but at least twice in the Cimicomorpha families, once in the are extremely important mechanically in finding the proper Cimicidae (containing the bed bug) including the small spot for meal suction [1]. sister group Polyctenidae (bat bugs), and in the Reduviidae Saliva is produced, sometimes copiously, during the (kissing bugs) from possible predacious or hemolymph- probing phase (the time between mouthpart contact with sucking ancestors [1]. Within the Reduviidae, it is possible the food substrate and the commencement of the meal) and that the genus Rhodnius (tribe Rhodnini) is monophyletic, throughout the meal [31, 32]. This saliva is ejected at the having evolved independently of the remaining triatomines tip of the maxillae by the salivary channel, which is built in (tribe Triatomini) [47–49]. The ancestral Cimicomorpha between the interdigitations of the two plates that form the dates back to the Triassic/Jurassic border, over 250 MYA [1]; maxillae [33]. Saliva helps probing and feeding physically by accordingly, the habit of blood or hemolymph feeding started 2 Psyche in this group well before mammals irradiated. Within these sequences into contigs (which represent full or near full- hematophagous bugs, blood is the only diet for all immature length mRNA), these can be compared by bioinformatic and adult stages. tools such as BLAST and rpsblast [69] to other proteins in To obtain blood in a fluid state, these bugs have to public databases (such as Swissprot, Gene Ontology [70], and counteract their host’s hemostasis, the physiologic process GenBank [71] protein data banks, and CDD, PFAM, SMART that prevents blood loss, which includes the triad of platelet and KOG [72], which are motif databases to be explored with aggregation, blood clotting, and vasoconstriction. Blood- the rpsblast tool) to identify closely related sequences and circulating platelets may be triggered to aggregate by var- functional motifs. Additional searches for signal sequences ious signals, including ADP from broken cells and also indicative of secretion [73], for transmembrane helices released by activated platelets, collagen from subendothelial [74], and for glycosylation sites [75] are also helpful to surfaces, thrombin (produced during blood clotting), and attempt functional classification of the protein. We are now thromboxane A2 (TXA2—produced by activated platelets). on the eve of another revolution, with the increase by Blood clotting may be initiated by activation of the intrinsic thousands of fold on the number of sequences that can pathway via activation of Factor XII or by activation of the be economically sequenced from these libraries, which will tissue factor pathway, both converging to the activation of allow identification of the lesser expressed (and possibly most Factor X to Xa, which activates prothrombin to thrombin potent) proteins. which in turn cleaves fibrinogen into fibrin, forming the So far, 12 sialotranscriptomes—all done with less than blood clot. Activated platelets produce the vasoconstrictor 3,000 sequenced clones per organism—have been reported TXA2 and also release stored serotonin and epinephrine, from Heteroptera, 11 of which are from blood-feeding both powerful vasoconstrictors. A single magic bullet cannot Cimicomorpha and one from the seed-feeding Oncopeltus properly destroy a redundant and complex obstacle such as fasciatus. Oncopeltus belongs to the Pentatomomorpha, the this; rather, a magic potion of several antagonists is required. closest group to Cimicomorpha [76](Table2). Among the Saliva of hematophagous arthropods also contains activ- Cimicomorpha sialotranscriptomes, only one derives from ities that interfere with the host’s immune and inflammatory Cimicidae (the bed bug Cimex lectularius); the remaining system in the form of immunomodulatory substances, par- are from Triatominae, encompassing four genera (Rhodnius, ticularly in ticks, which stay attached to their hosts for days or Triatoma, Dipetalogaster, and Panstrongylus), although some weeks, in contrast to minutes of host contact by bloodsucking of these transcriptomes have no proteins deposited in bugs. Saliva also contains antimicrobial compounds that public databases and too few expressed sequence tags (ESTs) might help to control bacterial growth in the meal, because publicly available. A few isolated protein sequences are also ejected saliva is reingested with the blood meal during blood available from GenBank, deriving mostly from predatory feeding. For more detailed reviews on host hemostasis and bugs. The publicly available proteins are displayed together immunity, see Francischetti et al. [56, 57]. in Additional File 1, which is a hyperlinked Excel spread- Salivary anticlotting compounds from bloodsucking sheet where the putative secreted proteins are organized in insects have been known to occur for nearly 100 years [58], one worksheet and the putative housekeeping proteins are while antiplatelet activity was first detected in the 1980s displayed in another worksheet. [59, 60] and vasodilators have only been described since the The secreted proteins can be classified in two major early 1990s [61]. In blood-feeding bugs, anticlotting agents groups, those belonging to ubiquitous protein families and from the saliva and crop of Rhodnius were described first those of unique status among the Hemiptera family, genus, by Hellman and Hawkins in 1965 [62], the first antiplatelet or even species level (Table 3). We will proceed to describe activity was reported in 1981 [59, 63], and Rhodnius salivary the protein families in the order shown on Table 3. vasodilator was reported in 1990 [64]. Anticomplement activities have also been found [65], as well as antihistamine, 3. Ubiquitous Protein Families antiserotonin [66], and antithromboxane [67] activities from Rhodnius saliva. An anesthetic was found in Triatoma 3.1. Enzymes infestans saliva in 1999 [68]. None of these earlier reports characterized the molecular nature of the compounds, most 3.1.1. Apyrase, 5 -Nucleotidase, and NUDIX Hydrolase. of these have been achieved in the past 20 years during the Apyrases are enzymes that can hydrolyze ATP and ADP to so-called “grind and find” period of discovery. Table 1 lists AMP [77–79]. Initially the existence of true apyrases was the molecularly characterized salivary components of blood- doubted, because they could originate from a mixture of feeding Hemiptera. enzymes such as adenylate kinase and ATPases; however, their real intracellular existence in the potato was shown later 2. On Sialomes [79, 80] and its function in carbohydrate anabolism and in the promotion of glycosyltransferases was only much later In the past 10 years, a new method to unveil the salivary discovered, as indicated [81, 82]. The role of extracellular potion of hematophagous insects has been practiced in the apyrases on preventing platelet aggregation was demon- form of decoding their sialotranscriptomes (from the Greek, strated for the first time in Rhodnius saliva [63, 83, 84] sialo = saliva), achieved by random sequencing of 500–2,000 and later shown in the saliva of mosquitoes [85–87] and in cDNA clones originating from polyA-enriched RNA from the vascular endothelium [88–90]. The activity from Cimex the salivary glands of these animals. After assembly of these lectularius was purified and cloned, revealing a new type of Psyche 3
Table 1: Molecularly and functionally characterized salivary components of bloodfeeding Hemiptera.
Name Family Insect Activity Notes Reference Prolixin Nitrophorin Rhodnius prolixus Anticlotting FXa inhibitor [2] RPAI Lipocalin R. prolixus Antiplatelet Binds ADP [3, 4] Binds histamine, carrier Nitrophorins Nitrophorin R. prolixus Antihistamine [5, 6] of NO BABP Nitrophorin R. prolixus Antiserotonin Binds serotonin [7] Inositol phosphatase Inositol phosphatase R. prolixus Inositol phosphatase Unknown function [8] Lysophosphatidylcholine Lipid R. prolixus Antihemostatic [9] R. prolixus, Cimex Vasodilatory, NO Inorganic gas Activates guanylate cyclase [10, 11] lectularius antiplatelet Apyrase 5 -nucleotidase Triatoma infestans Antiplatelet Destroys ADP [12, 13] Antiplatelet, Triplatin Lipocalin T. infestans TXA binder [14, 15] vasodilator 2 Triafestin Lipocalin T. infestans Anticlotting, antipain Inhibits FXII activation [16] Trialysin Trialysin T. infestans Antimicrobial Pore forming [17, 18] Collagen inhibitor Pallidipin Lipocalin T. pallidipennis Antiplatelet [19, 20] (possible TXA2 binder) Triabin Lipocalin T. pallidipennis Anticlotting Anti-thrombin [21–23] Procalin Lipocalin T. protracta Allergen Function unknown [24] Antiplatelet, Dipetalodipin Lipocalin Dipetalogaster maxima TXA binder [25] vasodilator 2 Apyrase Cimex apyrase Cimex lectularius Antiplatelet Destroys ADP [26] Antiplatelet, Nitrophorin Inositol phosphatase C. lectularius Carrier of NO [27–29] vasodilator Fibrinolytic enzyme Serine proteinase Panstrongylus megistus Anticlotting [30]
Table 2: Salivary transcriptomes of Hemiptera/Heteroptera.
Organism Number of ESTs on DBEST Number of derived proteins in GenBank Reference Rhodnius prolixus 1,439 56 [36] R. brethesi 55 0 [37] R. robustus 121 0 [37] Triatoma infestans 1,738 167 [38] Triatoma brasiliensis 2,109 28 [39] Triatoma matogrossensis 2,230 196 [40] Triatoma rubida 1,850 93 [41] Triatoma dimidiata 53 53 [42] Dipetalogaster maxima 2,671 66 [43] Panstrongylus megistus 45 0 [44] Cimex lectularius 1,969 102 [45] Oncopeltus fasciatus 1,115 37 [46]
enzyme that is ubiquitous in nature [26, 91, 92]. That for in the case of diadenosine nucleotides, which are known T. infestans, though, was found to belong to a completely agonists of platelet aggregation and inflammation [95–98]. different family, that of the 5 -nucleotidase family of enzymes C. lectularius sialotranscriptomes presents clear evidence of [12]. Interestingly, sand flies [93] express salivary apyrases such enzymes, but the activity in salivary homogenates was of the Cimex type, while mosquito salivary apyrases belong never studied. to the 5 -nucleotidase family [87, 94], clear examples of Lacking in these Heteroptera sialotranscriptomes are convergent evolution. additional nucleotide-acting enzymes, such as endonucle- Nudix hydrolases or bis(5 -nucleosidyl)-tetraphosphata- ases, found in mosquitoes and sand flies [99–101], and ses (EC: 3.6.1.17) are enzymes that hydrolyze nucleotides adenosine deaminase, found also in mosquito and some, but joined by their phosphate groups such as AP4A or AP5A not all, sand flies [102–104]. 4 Psyche
Table 3: Classification of the protein families relevant to secreted products in the Hemiptera/Heteroptera.
Classification No. of proteins Genera found1 Function characterized?2 References Ubiquitous protein families Enzymes Cimex apyrase 1 C, R(?) Y [26, 50] 5 -nucleotidase 6 T Y [12, 13, 51] Cimex NUDIX hydrolase 3 C Secreted esterase 5 C, T Inositol phosphate phosphatases including 24 C, T, R Y/N [8, 27, 28] Cimex nitrophorins Serine proteases 17 C, T Y/N [30] Chitinase 1 O Other enzymes 4 T, O Protease inhibitor domains Kazal domain containing proteins 13 T Serpin 1 C Pacifastin-related peptide 1 O Cystatin 5 O [2, 4–7, 11, 14, 15, 19– Lipocalins 331 T, R Y/N 21, 23, 43, 52–55] SalivaryOBP 19 C,T,R Salivary antigen 5 family 22 C, T, R Triatoma dimidiata lectin 2 T Immunity related Lysozyme 4 C Defensin 1 T Histidine-rich peptide 1 T Immune-related conserved insect protein 1 T Arthropod or insect specific families Cuticle-like proteins and conserved mucins 5 T Conserved insect secreted protein family 6 C, T, O Mys2 family 3 R, T Cimex-Triatoma family 3 C, T Other individual proteins of conserved insect 4T families Hemiptera specific families Mys3/hemolysin-like family 16 T, R, O Triatoma-specific families Trialysin 8 T Y [17, 18] Short trialysin 6 T Triatoma matogrossensis family 2 T Triatoma matogrossensis family 2 2 T Orphan Triatoma proteins 19 T Rhodnius-specific families Orphan Rhodnius proteins, include 3R low-complexity proteins Cimex-specific proteins Cimex mucin family 2 C Orphan Cimex proteins 1 C Psyche 5
Table 3: Continued. Classification No. of proteins Genera found1 Function characterized?2 References Oncopeltus-specific families Oncopeltus family 3 O Oncopeltus family 2 2 O Orphan Oncopeltus protein 12 O Total 559 1 C: Cimex;T:Triatoma/Dipetalogaster/Panstrongylus;R:Rhodnius;O:Oncopeltus. 2Y: yes; Y/N: characterization of a few or a single member of the family.
3.1.2. Acetylcholinesterases. Four well-expressed and closely and ticks, being thus uniquely from Cimicomorpha blood related isoforms of a typical acetylcholinesterase enzyme feeders. were found in the sialotranscriptome of C. lectularius [45]. A single transcript from the same family was also found in Tri- 3.1.4. Serine Proteases. Serine proteases are commonly found atoma matogrossensis. Although most acetylcholinesterases in the sialotranscriptomes of insects and ticks, as well as in are extracellular membrane-bound enzymes by virtue of those of Heteroptera. An unusual serine protease activity in a glycophosphatidyl-inositol membrane anchor in their the saliva of T. infestans has been noted before, but only a par- carboxy termini, these Cimicomorpha enzymes lack this tial enzyme purification of the enzyme, named triapsin, was terminal region, and thus these enzymes are secreted. The achieved [107]. Within the bloodsucking Heteroptera, only role of these enzymes in blood feeding is not yet apparent. one Panstrongylus megistus sequence has been molecularly characterized as a fibrinolytic enzyme [30]. Additional File 3.1.3. Inositol Triphosphate Phosphatases (IPPase) Includ- 1 shows such proteins from Cimicomorpha, including plant- ing Cimex Nitrophorin. This family of proteins has been feeding bugs such as Lygus lineolaris, Lygus hesperus, and Cre- found ubiquitously in the sialomes of bloodsucking Cim- ontiades dilutus [108–110]. The phylogram of these enzymes icomorpha, including the well-characterized enzyme from (Figure 2) shows two well-defined clades, one containing R. prolixus [8] and the C. lectularius nitrophorin [27–29], a most of the Lygus sequences, but also two T. matogrossensis protein found associated with a heme moiety and a carrier and one T. brasiliensis sequence, within a clade of 86% and stabilizer of nitric oxide (NO), a very reactive gaseous bootstrap support, suggesting a common ancestral salivary substance that is also a potent vasodilator and platelet aggre- serine protease for plant- and blood-feeding Cimicomorpha. gation inhibitor. While the function of Cimex nitrophorin The fibrinolytic enzyme of Panstrongylus shares a strongly is without question, the function of an extracellular inositol supported clade with two other T. matogrossensis sequences, phosphatase is puzzling, because these inositol phosphates which are probable orthologs of the Panstrongylus gene. The are intracellular and not available to an extracellular enzyme. Cimex sequence appears as an outlier to the group. Rhodnius Indeed, it appears fitting that inositol polyphosphates should sialotranscriptomes have not revealed proteases, and its saliva be hydrolyzed, because they perform a proplatelet aggre- does not hydrolyze the substrates used in the characterization gation function as well as proinflammatory and immune- of the T. infestans triapsin (Ribeiro, unpublished). enhancing roles in leukocytes [105, 106]. Perhaps the enzyme may reach the intracellular pool by some not yet understood mechanism. On the other hand, association of heme with 3.1.5. Other Enzymes. A chitinase and a lipase were found in inositol phosphatases seen in the case of Cimex nitrophorins Oncopeltus, while T. matogrossensis displayed a salivary phos- is not at all common, being unique to these proteins; inves- pholipase and a metalloprotease. The precise role of these tigation of the amino acids that are associated with heme enzymes is unknown. Salivary metalloproteases in ticks binding does not reveal similarities to other IPPases from have been associated with fibrinolytic and antiangiogenic either vertebrates or invertebrates (Ribeiro, unpublished). activities [111, 112], while the Oncopeltus enzyme may be The phylogram of the IPPase sequences found in Addi- associated with digestive or antifungal functions. tional File 1 (Figure 1) shows the Cimex nitrophorins con- tained within a strong clade with 100% bootstrap support 3.2. Protease Inhibitor Domains and constituted by at least three subclades representing at least three genes expressing these NO transporters, plus 3.2.1. Kazal Domain-Containing Peptides. The Kazal domain alleles or other genes. Cimex has two additional sequences occurs in many protease inhibitors, and its structure was outside the nitrophorin clade and near the IPPase clade of the first determined for the proteinase inhibitor IIA from bull remaining triatomines. It is thus interesting that both Cimex seminal plasma [113]. The sialotranscriptome of members and triatomines have a common IPPase in their sialome, even of South American Triatoma (T. infestans, T. matogrossensis, though we have no idea of their function. IPPases have not and T. brasiliensis) but not North American T. dimidiata or T. been found in any other transcriptome so far done, including rubida, nor any other sialotranscriptome of Cimicomorpha, those of mosquitoes, sand flies, biting midges, black flies, abounds with transcripts coding for proteins containing this 6 Psyche
82 CIMLEC_263173211 CIMLEC_263173166 CIMLEC_263173300 100 CIMLEC_263173215 69 CIMLEC_263173444 98 CIMLEC_263173207 99 CIMLEC_263173222 Cimex nitrophorins 100 82 CIMLEC_3388165 CIMLEC_263173256 CIMLEC_263173380 CIMLEC_263173328 95 100 CIMLEC_263173332 88 82 CIMLEC_263173281 CIMLEC_263173339 CIMLEC_263173336 TRIMAT_307095198 RHOPRO_90101342 99 TRIINF_149689206 TRIINF_149689170 97 IPPases TRIMAT_307095030 69 TRIMAT_307095032 61 TRIMAT_307095034 49 TRIDIM_270046240 37 0.2 99 TRIDIM_270046238 Figure 1: Phylogram of the inositol triphosphate phosphatase family of Cimicomorpha. The sequences are named with the first 3 letters of the genus name, followed by the first 3 letters of the species name, followed by their GenBank GI accession number. The sequences were aligned by Clustal, and the neighbor-joining bootstrapped phylogram was obtained with the MEGA package with 10,000 iterations, Poisson model of amino acid substitution and pairwise amino acid comparisons using the gamma rate of amino acid substitution (gamma parameter = 1). The numbers at the nodes are the percent bootstrap support. The line at the base indicates the rate of amino acid substitution per site.
27 LYGLIN_18034141 26 LYGLIN_33772619 100 LYGLIN_33772615 LYGLIN_33772613 54 LYGHES_14599860 Lygus/Triatoma TRIMAT_307095022 86 99 TRIBRA_111379925 TRIMAT_307095018 30 94 CREDIL_16304416 LYGLIN_33772617 TRIMAT_307095024 59 98 TRIINF_149689028 PANMEG_295315341 TRIMAT_307095026 99 Fibrinolytic 81 TRIMAT_307095020 CIMLEC_263173321
0.2 Figure 2: Phylogram of the salivary serine proteases of Cimicomorpha. See the legend of Figure 1 for more details.
domain; however, none have been so far characterized func- lipocalins named prolixin S and triabin [2, 21, 118]. Kazal- tionally. In Rhodnius, Triatoma, and Dipetalogaster, the crop type peptides can function as antimicrobials by inhibiting antithrombin has been characterized as a protein containing microbial exoproteases essential for their survival [119, 120] two such domains [114–117], but salivary anticlotting of and can also work as vasodilators, as in the case of a tabanid Rhodnius and Triatoma has been shown to be different salivary protein named vasotab, which is suspected to modify Psyche 7 ion channels [121]. These functions should be taken into tuning its function in relationship to its target. For example, consideration in functional assays of the recombinant Kazal a bug feeding on a bird may have “ideal” anticlotting, but peptides. if ecologic changes appear and the bug shifts to another host, this anticlotting may still work but have some room for ffi 3.2.2. Serpin. The serine protease inhibitor (serpin) family is improvement (e.g., by increasing its a nity to the specific ubiquitous in nature, functioning mostly as endogenous reg- thrombin). Second, any protein injected into the skin of a ulators of proteolytic cascades such as inhibiting thrombin in vertebrate is capable of eliciting an immune reaction, which vertebrates (plasmatic antithrombin 3) or regulating phenol may lead to defensive host behavior following mast cell oxidase activation cascades in invertebrates [122, 123]. The degranulation or complement-mediated local inflammation, salivary anticlotting proteins of Aedes mosquitoes (but not leading to interruption of the meal or killing of the insect. those of anopheline mosquitoes) are members of this family This may lead to a scenario of balanced polymorphism, with [124, 125]. A single sequence of this family, derived from four the least common epitope being the best one to have, thus ff ESTs, was found in the sialotranscriptome of C. lectularius. multiplying the number of di erent alleles in a population Its target is still unknown. that are selected to have the same optimal function but the least common antigenicity. Host immune pressure can also lead to gene obsolescence, creating a niche for cooption 3.2.3. Pacifastin and Cystatin. Proteins containing these (exaptation) of new genes, including horizontal transfer domains were only found in the sialotranscriptome of [138], which may substitute for the lost function and thus Oncopeltus. Pacifastins are typical serine protease inhibitors may explain the appearance of novel salivary genes in related of insects and crustaceans [126], while cystatins are ubiq- organisms [139]. uitous proteins typically inhibiting cysteine proteases [127]. Lipocalin functions in triatomines are multiple and Although a single EST was found coding for the pacifastin linked to their unique barrel when working as kratagonists peptide, five well-expressed cystatins were identified in (from the Greek kratos = seize) [140], which are binders Oncopeltus. The targets of these peptides are unknown, but of relatively small agonists such as biogenic amines, TXA2, it was suggested that the salivary cystatins may prevent plant leukotrienes, or ADP, or carrying the heme that carries NO apoptosis induced by cysteine proteases [46, 128, 129]. Tick in Rhodnius nitrophorins, or functions linked to their side sialomes have revealed cystatins that were shown to inhibit chains when they work as anticlotting agents such as triabin inflammation and maturation of dendritic cells in their hosts (for references for these functions, see Table 1). Uniquely, the [130]. protein nitrophorin 2 from R. prolixus has three functions: (i) it carries NO, (ii) it binds histamine, and (iii) it is an inhibitor 3.3. Lipocalins. The term lipocalin literally means a cup of of the activation of Factor X [5, 141]. Notice that contrary to lipid, as these proteins form a barrel with a hydrophobic their names as “lipid cups,” many of these lipocalin ligands interior cavity that is suitable to transport lipids and other are well charged and not hydrophobic, such as biogenic hydrophobic compounds in an aqueous milieu [131–133]. amines and ADP. The functions of the salivary lipocalins in There is virtually no sequence conservation in the family, ticks are similarly associated with their kratagonist activity which is recognized by its typical 3D structure composed of toward biogenic amines or arachidonic acid derivatives, or as arepeated+1topologyβ-barrel. This protein family is by inhibitors of complement activation [142–148]. far the most abundant in sialotranscriptomes of triatomine A phylogram of the triatomine lipocalins, although a bit bugs (see review [132]) but remarkably absent in Cimex overwhelming in size, presents a bird’s-eye view of the several and Oncopeltus; however, it was also abundantly recruited distinct families arranged mostly in robust clades (Figure 3 in tick sialomes [56], another case of convergent evolution. and Additional File 2). Most clades have not a single member Additional File 1 provides for 331 lipocalins, which is more that has been analyzed functionally (marked with Roman than half of all putative secreted proteins listed in this numerals in Figure 3), including the clade containing the work. Several of these proteins may be alleles of the same Triatoma protracta antigen procalin; accordingly there are gene. The sheer size of the family in individual species is eight clades that have no known function. Additional File 2 is indicative of gene duplication events that might have had provided for high-resolution display of the sequences, which an impact during the evolution of blood-feeding [134– have their NCBI accession numbers for sequence retrieval. 136]. Following gene duplication—by retrotransposition A few details deserve some comments with respect to the or more commonly by forming tandem repeats due to phylogram. (i) The clade named Pal-Tri-Dip contains the transposable element recombination—the new genes can Triatoma proteins pallidipin, triplatin, and the Dipetalogaster lead to an increased transcript load in a particular organ or protein dipetalodipin, which are platelet inhibitors possibly tissue. If this augmented expression increases fitness (e.g., all due to being TXA2 kratagonists as demonstrated for helps the bug to feed), the gene will persist; otherwise, triplatin and dipetalodipin [25], thus indicating the conser- it will evolve to be a pseudogene [137]. Once genes are vation of this function among two different genera. (ii) Most duplicated and fitness is increased by the duplication, these Rhodnius lipocalins cluster in two clades, one containing are free to evolve independently and to diverge from each all the known NO carriers, named nitrophorins (NP) and other by acquisition of novel functions. Salivary genes of the other containing the adenosine nucleotide kratago- bloodsucking arthropods are under selection by two different nists named RPAI (Rhodnius platelet aggregation inhibitor). processes. First, the gene can evolve in the direction of fine (iii) The Rhodnius biogenic amine-binding protein (BABP) 8 Psyche
Triafestin
T 2
- 1
N -
N
A NT I N I
D T T
S N DA S
E
U N E
F B F
A
A BU I A I
_
A R T
6 R 4
2 _ T N
0 T 4 8 0 _ 8 A 9 _ 8 6 8 7 8 6
4 6 D 4 6 4 0 7 0 3
9 0 9 0 9 4 N 9 1 1 9 2
0 9 17 0 9 3 0 8 9 9 U I 8 4 8 7 8 9 9 7 1 7 6 R 2 8 6 8 5 B
0 6 4 0 9 2 0 R 9 0 9 NP-BABP H 9 4 A
3 02 9 9 0 3 8 0 3 4 5 8 48 0 O R H 4 9 _ _ 4 5 0 _ 6 2 0 _ 1 5 9 9 7 4 O R 1 0 H 1 7 6 2 P T 9 R T II T _ 1 4 0 2 _ 7 5 2 15 0 4 6 R O P H _ 4
A H A F _ 1 7 8 A 0 3 5 F _ 3 0 4 F 1 0 O P R O O F _ 3 0 8 5 M N M F _ 0 0 R M N _ I I I N 3 42 6 I 2 _ R O P I F _ 0 I 0 P I N T 7 36 I F I N 5 HO I _ 5 4 9 1 R R O R R I T 6 R _ R I N 2 7 8 R A I 5 3 4 R I N T 5 1 TRIMAT_307095180 1 T R 4 T I _ 2 2 H T I A _ O R R O T 3 5 8 3 T I 5 R M A _ 9 0 O PRO 3 T 4 R T R I M _ 2 H 8 _ 7 _ M 3 44 4 2 T R I I 5 3 M M 4 P 9 4 3 T R B _ O H 5 I I 9 2 6 0 T 5 1 R 5 R D 5 8 R 3 2 3 B _3 0 6 8 PR O 9 7 T I U 3 R 1 5 5 R D 5 5 H _ 0 62_ T I X 7 4 1 O 2 R U _ P 9 R 3 6 H 1 _ 4 1 T 0 0 9 9 T O 8 I A R 1 5 R B 5 O O RO _ PR 9 8 T R _ 0 8 4 5 6 3 P R I 2 N H 1 0 T M U B 9 P _ 7 7 N 6 _ 7 6 9 A R 57 _ 6 T R 0 49 R O 3 5 4 6 P R U T 2 9 8 8 R _ O N P 9 I I 7 9 D H O 35 7 _ 1 T _ 4 4 7 P O 6 5 LI R A 0 0 N 6 NP P - 99 7 D R I 1 8 8 O R _ 6 7 3 M 3 7 9 RPAI _ 4 1 0 - 9 T M I _ 4 U 2 X R 0 0 P O 6 3 I _ 3 6 8 2 _ F 3 7 9 88 B 4 I - T R D T R _ 3 4 65 N N 3 I 0 4 8 A 6 _ N _ III O 3 5 1 T R I 30 7 9 5 _ 1 6 N P -S I T 3 1 9 _ T _ 0 0 1 1 _ 8 - R MA A 9 T 2 5 0 _ N P 2 T 3 7 R 1 6 9 N - T M _ 0 8 81 0 7 6 P RI I A T 9 8 3 P 4 3 6 8 9 I 6 5 - T 9 1 2 R M _ 5 R M _ 9 6 67 _ - 1 I AT 4 6 1 A 8 N 4 9 T T R ID N R 1 98 4 9 H T 0 P A IM A 4 R I 4 _ P T _ 4 0 4 R O M _ N - R M F 1 0 9 2 H 3 7 -4 7 I 0 O H P _ P T _ 7 1 0 _ 0 B 9 R IN F 2 7 9 P R O R 2 7 B -1 3 T I _ 0 8 9 R H PR O 7 0 A R IN 3 6 1 A 0.5 I M _ 9 8 9 O O _ 0 B T I 9 8 8 3 3 4 P R D T 4 1 6 _ P O_ 89 I 1 6 6 1 R 35 8 T A 9 9 R 1 6 R _ 8 8 5 O 3 0 IM 4 6 4 6 H 7 3 1 6 9 9 T F 1 8 2 _ 8 1 9 R N _ 9 9 R O 7 3 5 6 9 I 0 8 P 1 T I F 14 7 4 R H 2 3 8 7 R 0 9 2 H O R 5 5 3 IN _ 0 4 O R O _ 1 6 T I F 3 1 P R 7 R _ 7 1 P H _ 8 9 9 IN T 0 9 9 O R P 6 9 T 3 8 1 R O 3 6 8 A _ 6 7 O P 35 P 9 9 RI 9 3 R _ A 1 T IM T 6 0 _ 3 18 9 A 4 2 2 T 1 O 3 I- R 1 6 7 3 R 5 _ 5 7 1 T IM _ 6 2 7 3 1 1 F 11 2 9 IR 2 3 8 1 R N _ 6 9 T U 72 5 6 9 T II A 1 7 0 R 1 9 1 1 I B 7 7 R R _ 3 9 R _ _ 8 5 9 T B 1 4 T 6 0 5 I A 1 9 4 T R U 3 R 7 0 R 1 4 N B 5 1 R 0 IR 5 P 1 T B _ 7 3 A TR _ 4 A 1 I A 0 28 0 D U 3 28 I R R 3 5 N B 5 -2 T _ 4 3 U T IR _3 5 31 IB T 5 8 B R T U 4 R A 5 2 6 A IRU R 5 2 2 3 4 4 _ B 5 8 5 8 T M _ 5 3 I _3 3 7 I B T T B R 4 2 5 R 5 8 S N _ T UB 5 2 6 T U 3 42 42 O A 3 R 5 8 9 IR _ 5 3 D 5 IR _ 4 30 9 B 5 8 M N T 5 U 3 2 R U 3 2 8_ N 4 5 8 8 9 T R _ 4 U A 2 B 5 3 89 2 I 5 6 B 8 _ 4 1 R B 5 82 A D 2 3 2 0 U 3 2 _ N T 5 5 8 0 5 T R _ 6 U R 8 5 3 9 94 I 54 2 B IR _ 4 1 9 4 R B 5 8 A IV A 2 4 7 T U 4 _ T U B 83 R _3 9 4 R B U 5 I B 70 0 I _ N 2 7 R U 0 9 R 3 D 2 74 T 3 94 T U 5 A IR _ 0 8 R B 54 N 78 R T 7 82 IR _ 2 99 T A 0 4 U 35 82 T IM _3 9 T B 5 8 R T 70 8 R _ 42 8 T A 0 18 IR 3 8 IM 3 6 U 55 3 7 7 1.5 _ 4 T B 2 8 7 R T 0 4 RI _ 42 4 T A 0 7 RU 3 8 IM 27 61 55 3 R _ 4 TR B 4 16 T IM 00 72 I _3 28 9 0 D 7 1 R 5 2 9 6 I _2 6 T UB 54 6 TR M 04 R _ 2 0 I 70 28 I 3 8 5 ID 2 2 R 5 2 1 R _ 6 U 54 54 T M 04 TR B_ 28 I 0 76 T I 3 2 ID 27 1 R RU 55 5 7 TR _ 46 IR T B 42 0 7 IM 00 U R _3 8 ID 7 66 B_ IR 5 25 R _2 91 35 U 54 2 99 T IM 8 54 B_ 28 D 96 4 28 35 26 0 RI 14 16 24 54 2 7 T F_ 89 8 2 IN 96 0 TR _A 82 RI 4 96 IR BU 86 T _1 94 UB N 2 NF 70 T _ DA 71 II 30 64 RI 35 N TR _ 48 RUB 54 T AT 09 _ 28 IM 07 6 TR 35 25 TR _3 18 IR 542 6 AT 95 UB 8 9 9 IM 70 _ 2 9 9 R 0 TR 35 84 T _3 40 IR 54 AT 949 U 28 57 IM 0 B_ 26 R 307 6 TR 3554 4 T T_ 93 IRU 2 MA 094 B_ 826 RI 07 T 35 6 T T_3 38 TRI RIR 54 72 A 949 PR UB 283 RIM 70 O_1 _35 32 99 T 30 542 542 72 AT_ 890 64 8 88 6 IM 98 13 33 0 TR 98 _PR 4 _14 DIP O NF 176 MA CAL 2.5 99 RII 89 X_ IN T 496 344 _1 2 DIP 190 IINF 487 MAX 586 TR 709 _34 0 _30 T 419 9 AT 850 RID 05 RIM 094 IM_ 92 T 307 2700 AT_ 2 TR 461 RIM 483 IDIM 62 98 99 T 709 _27 T_30 0046 53 IMA 34 TRID 156 TR 0948 IM_2 _307 7004 97 IMAT TR 6160 TR 8911 IDIM 4989 _2700 F_1 4615 TRIIN 8 TRID 8 68921 IM_27 3 _149 004 90 78 INF 6244 TRI 90 TRIDI 9 6890 M_270 7 _149 04617 TRIINF TR 8 8926 IDIM_2 _14989 700461 TRIINF 70 08 TRIDIM 69 96892 _2700 85 INF_14 46168 3 TRI 6 198 TRIDIM 93 _270046 _2700461 TRIDIM 64 99
Procalin 92 96 TRIDIM_ _2700461 270046166 TRIDIM 1 DIPMAX O_3351868 _344190587 RHOPR 99 DIPMAX_3441 3518677 90543_ABUNDA RHOPRO_3 NT 87 3518683 DIPMAX_344190596 70 RHOPRO_3 18719_NP-3B DIPMAX_344190546 85 RHOPRO_335 99 DIPMAX 3 41 RHOPRO_33518691 _ 4 90557 92 DIPMAX_344190595_ABUNDANT 63 53 61 TRIMAT_307094880 60 T DIPMAX_344190585 RIMAT_307094836 TRIINF_ DIPMAX_344190575 149689034 0547 TRIMAT_30 DIPMA X_34419 99 7094868 V T 4190560 RIINF_1496 DIPMAX_34 89032 ANT 83 TRIINF_ 4_ABUND 149689116 _34419054 93 DIPMAX 4 TRIM 4419055 76 AT_3070 IPMAX_3 95178 D TR 190572 IMAT_3 AX_344 54 0709486 DIPM T 2 DANT 1 RIINF ABUN 6 _14968 90584_ 9174 _3441 NT 99 TRID IPMAX UNDA IM_27 D 74_AB 00462 41905 4 TR 46 X_34 9056 IDIM_2 DIPMA _3441 54 700 MAX T 46214 DIP 0573 RIDIM 4419 _270 AX_3 T 0462 IPM 53 99 99 RID 18 D 1905 IM_ 344 2700 AX_ 6 79 TR 4625 DIPM 055 IDIM 0 4419 _270 X_3 T 04 MA 567 87 RID 623 DIP 190 IM_ 4 VI 44 6 270 X_3 0 5 2 TR 046 A 055 9 IDI 212 DIPM 419 M_2 _34 D 700 AX 588 IPM 462 IPM 190 AX 10 D 44 3 _34 X_3 1 9 TR 41 MA 988 94 IDIM 905 DIP 137 _2 58 _11 1 RH 700 RA 990 OP 462 RIB 137 RO 30 T 11 3 TR _33 A_ 718 IDI 51 IBR 26 99 M_ 868 TR 16 0 TR 270 5 A_1 06 IB 04 BR 689 RA 618 RI 49 8 T _11 6 T _1 05 59 RIB 1 NF 89 R 379 II 6 T A_ 90 TR 149 54 7 R 11 7 F_ 90 2 IM 13 IIN 68 AT 79 R 49 8 TRI _3 88 T _1 90 M 07 5_A F 98 AT 09 BU IIN 98 2 8 TR _ 48 N TR 14 5 6 96 I 30 96 D F_ 90 MA 70 AN IN 68 T T 94 T RI 49 62 R _3 89 T _1 49 IM 07 8 F 9 T AT 09 IIN 70 3 R _ 49 TR 30 91 IM 30 02 T_ 79 4 A 70 A 13 5 9 TR T_ 94 IM 11 91 97 IM 30 83 TR _ 79 T A 70 0 A 3 T 5 R T_ 9 BR 11 AN 6 II 3 49 RI _1 D N 07 00 T A N 8 T F 0 R U 85 1 RI _1 94 IB AB 71 IN 09 83 R _ 6 TR F_ 24 8 T 81 62 87 3 0 1 1 1 IIN 4 37 67 _1 67 T T F 42 1 2 A 2 N R _ 16 _T 6 R 6 A IM 1 5 R 11 B 1 D 66 49 4 IP I _1 N 6 T A 6 L A_ TR A U 7 R T_ 8 A R R B 8 3 IIN 3 91 T IB IB A 94 T 5 T F_ 0 9 IN TR R _ 0 N R 70 6 -1 T 44 7 A 99 IIN 1 9 9 30 D 9 T F 49 4 4 _ N 3 7 R _1 6 93 09 T U 89 6 II 8 2 7 A B 9 4 T N 09 90 30 IM A 7 0 99 4 R F 2 7 _ R _ 3 92 6 I _3 4 8 T T 83 11 4 T IN 4 03 _A A 8 1 9 8 R F 4 7 B IM 9 _ 0 91 7 I _ 2 3 U R 37 A 07 4 6 TRIM INF 14 16 _T N T 1 R 3 9 56 9 5 R D 11 IB _ 0 8 _ 6 6 I A _ T 7 4 T 14 8 P N A TR A 0 9 6 8 RI A 9 9 LA T M _3 0 4 5 1 T_ 6 05 R I T 7 8 7 T M 8 0 T IB R 0 4 2 7 R A 3 9 IN R T A 3 9 2 9 0 0 T _ 0 9 IMAT T 7 2 -2 IM T 7 4 8 T _ 0 0 R A 9 2 7 R 3 9 T 30 0 7 7 5 6 IM 0 4 M _ 9 9 T _ 7 9 I T 7 4 4 9 1 R AT 3 0 1 R A 0 9 2 4 9 4 T 3 0 9 5 T II 0 4 M _ 7 4 T 4 N _ 7 8 I T 0 7 R F 3 0 6 R A 3 9 N I 0 9 0 T _ 0 A T P _ 7 4 M 7 2 9 1 I T 0 D 8 R A 4 0 9 1 I L 9 4 R A 3 N 1 T D _ 96 2 T _ U 5 6 R I 4 IM T B 9 2 M 3 8 8 R A 0 9 D ID 88 9 8 A 7 4 2 I _ 0 4 T M _ 9 5 P IM 2 3 6 I 6 0 6 DI M 7 5 2 R 1 3 0 1 4 _ 0 9 T 9 _ 7 2 6 P A 270 0 _ 4 T 0 9 D M X_ 4 P 9 3 4 4 I A _ 4 4 9 0 4 P 6 A 0 3 9 9 T A 0 2 L 7 IM T 0 8 7 0 M X 3 4 2 _ 1 9 RI 4 LI 0 A 7 4 7 7 62 4 R 6 DI A _3 4 3 F 9 5 7 D T M 0 8 7 9 6 RU X 1 _ I N 3 0 9 22 I I 9 9 9 3 P 4 9 P T R I _ 7 7 9 D _ 4 A 0 8 2 8 3 M B 3 0 I T R T 3 9 I 19 5 N T 3 9 8 D P A _ 4 IM A _ 1 7 1 0 4 6 -2 1 0 7 I M 3 0 T 3 6 8 3 PM X 1 1 R M 5 8 D 5 5 I 1 4 8 A _ 9 T A _ 1 6 I 5 9 R 11 0 6 2 T P X 3 4 0 0 M 6 2 T I A 1 0 4 2 6 A _ 4 2 5 _ 6 RI M R R _ 7 0 6 6 4 5 TR X 3 4 8 6 D 8 6 1 9 T B A 2 0 4 3 0 M A _ 4 37 I I _ 6 1 T X 4 9 P 7 2 6 3 R 0 8 7 I 0 R 4 9 A 1 2 E B M 2 0 2 3 4 R B _ 4 5 T I I 6 1 T _ 7 4 8 8 T 3 4 9 T 2 R I R 5 3 9 I 0 A R D M 2 5 2 9 T N _ 4 1 5 I 7 5 A T I _ 5 4 8 2 7 I 3 4 9 5 L R 2 0 2 R I F _ 4 D 3 5 2 6 T N 0 1 0 O T I M 4 8 4 8 I _ 1 9 I _ 5 3 R M 70 9 5 D Pa1-Tri-Dip T F R 5 2 9 7 1 1 0 6 B 3 8 8 5 I I T ID 5 4 1 T R M A _ 4 1 9 8 PI _ 2 0 7 5 U 3 5 6 2 I 1 9 3 4 R B 4 2 8 T R B A T 48 N R _ 5 4 0 0 49 6 7 9 T I 5 6 2 R I _ U 3 8 5 T R T 8 9 B 2 9 I 5 5 4 5 2 N 3 R _ 9 8 1 R 8 R 00 9 9 T I A _ 9 9 0 I 3 8 8 I 0 0 8 8 9 T 7 8 T R N F 3 0 1 _ 0 4 7 I _1 7 R RU 2 6 8 3 I _ 0 8 8 9 I UB 7 9 8 - T R I N F 0 T B 9 2 I 1 7 8 0 _ 9 6 T R 1 9 R R 2 0 N - I N F _ 1 I U 4 7 4 9 I 4 0 8 I 4 T 4 T M _ 7 - R N 3 6 6 6 2 I F _ I 1 3 N 1 T 9 9 R R 1 B I - 7 R I 4 267 8 I 0 9 I N 1 M _ 1 N 6 T F _ 8 4 T D 5 I R I 4 7 I 3 A B T I N 4 I N 2 94 R 1 2 R F _ 1 9 8 F I T I 0 _ 3 I 0 I 2 B R N 9 A 6 D 1 23 4 T I 4 8 4 T R 3 I 4 F _ 1 1 I N R B 5 T R I N 1 T 2 8 I _ A 2 T I F 9 8 0 T 6 5 T R I _ 14 4 9 I _ I T N 6 R 1 T R IN
A T A 5 0 I 9 6 D F
T R 8 2 A 4 D _ 9 1 T 1 9
T
T I B 1 T 5 N T R TR R O _ T R 9 4 T F 8 4 1 3 R I B _ 8 9 2
0 R 1 M 9 I 4 9
R R I M 8
R F
9 R
I R RI R
5 _ 8 5 R 8 A R T I T P I
0 9 1 I _ 8 R P 1 0 IM R 4 8 9 8 B 9 8 I N _ B 8 6 I 0 7 _ T 9 I
8 9 I A 1 I 8
0 I M M A R I P 7 D I 4 8
M 9 8 9
6 M
B M D A 9 D 1 D R 3 8 _ 9 4 9 3 6 F 4 0 T 7 8 _ 9 7 R 2 9 A T 6 9 8
N A A
O 2 3 9 7 R _ 4
8 A
I I A A I A 9 1 _ 2 9 6 _ 1 8 8 8 78 8
T 4 M M _
2 2 8 3 M T 9
H 3 14 U 09 T 1 X
_ 4 9 A 1 8
T X 9 2 T _ 1 9 7 1 3 1 1 _ 68 2 2 3 M _ 7 1
1 B 4 _ _ _ 4 9 R _ _ 8 _ 6 7
I _ 1 1 _ 9 1 M _ _ 0 1 2 1 3 2 F 0 1
I _ 1 2 2 3 9 8
3 2 8 1 2 3 5 3 1 3
D F 3 7 1 2 1 0 9 1 3 3
7 7 0 8
I _ 7 6 8 N F 4 6 8 _A 0
D 1 6 0 L_ I 4 0
_ 1 1 I N _ 7 7 1 _ 0 0 7 3
0 8 4 I 8 F 4 7
R I 6 7 2 7
N 4 9 A _ I 0 9 3
I A 0 0 0 0
T 0 9
2 1 1 2 09 6
0 T 1 _1 R I N 4
P AL L 9 9 6 I 4 4
4 9 0 TR R 1 R A 9 9
I 6 7 9 L T I 9 8 A R 4 0
P 6
6 6 4 1 T 9 0 5 48 1
7 4
R I B 0 4 A T R M 9 3
I 2
P 2 2 8 8
8 I 5 2 8 9
T I 5 8 R 48 T _
0 P 0
3 7 6 R 6 5 9
0 3 I 7 T R A
6
4 2 4 9 5
4 T 5 4 R TR 6 T B 4 T 1 U
_ N
F D
N I A I N
R T T
Triabin VII Figure 3: Phylogram of the salivary lipocalin family of triatomines. Clades containing members that have been functionally characterized are named according to these proteins. Other clades are named with Roman numerals. Except for the triafestin clade, other clades have >70% bootstrap support. The sequences are named with the first 3 letters of the genus name, followed by the first 3 letters of the species name, followed by their GenBank gi| accession number. When the protein has been functionally characterized, its name is also included after the NCBI number. Abundantly expressed proteins are also marked to indicate this fact. The numbers in the bar indicate the rate of amino acid substitution per site. For other details, see Figure 1 legend.
somewhat surprisingly clusters with the nitrophorins, but are exclusive of the genus Rhodnius. Indeed the abundance of BABP does not have a heme group and has higher affinity for these heme proteins in Rhodnius salivary glands makes these serotonin and norepinephrine, constituting a good example glands distinctively bright cherry red in color, as first pointed of gene duplication and divergence of function [7]. (iv) out by Wigglesworth nearly 70 years ago [149]. Triatoma Exceptionally, one T. matogrossensis and one T. dimidiata and Dipetalogaster glands are clear or of a very pale yellow protein sequence group with the NP-BABP clade with 99% color [150]. (v) Rhodnius lipocalins not belonging to the bootstrap support. The function of these proteins could lead NP-BABP and RPAI clades are scattered in the phylogram, to the original function of the Rhodnius nitrophorins, which including one sequence between the procalin and triabin Psyche 9 clades, one between the triabin and VI clades, and a group could function as an antimicrobial peptide, and a defensin is of four proteins between clades IV and V. None of these reported from T. infestans. The absence of commonly found Rhodnius proteins group within strong bootstrap support salivary antimicrobial peptides in triatomines suggests that to any of the Dipetalogaster- or Triatoma-containing clades. if this salivary function is present within these organisms, (vi) Finally, the procalin clade is very extensive and contains it may be encoded by lineage-specific gene families, one of many robust subclades, many of which are of single species, which (trialysin) will be reported further below. indicating possible recent events of gene duplication or extensive polymorphism. 4. Arthropod-Specific Families
3.4. Odorant/Pheromone-Binding Family (OBP). The OBP Several insect-specific families are further identified, none family, like the lipocalins, is specialized in carrying small functionally characterized, and most without domains pro- hydrophobic ligands in aqueous media [151, 152]. A mod- viding a clue for their function. These include proteins with ified version of the odorant-binding family of proteins is chitin-binding domains and cuticle-like homologs, which very abundant in the sialotranscriptomes of hematophagous may be associated with salivary ducts rather than a function Nematocera [138] and named as the D7 protein family. A few in the injected saliva. One conserved secreted insect protein mosquito proteins have been crystallized and functionally family of basic peptides having ∼100 amino acids after characterized, showing kratagonist activity toward biogenic signal peptide cleavage occurs in Cimex, Oncopeltus, and amines, TXA2, and leukotrienes, in addition to anticlotting Triatoma sialotranscriptomes. Homologs are found by blastp activity [153–156]. to the nonredundant (NR) protein database including a In hematophagous Cimicomorpha, members of the OBP venom protein from the wasp parasitoid Nasonia vitripennis family are found in Rhodnius, Triatoma, and Cimex but identifiedinaproteomicstudy[165]. Exceptionally, there are are particularly abundant in Cimex, with two OBP proteins also homologs to proteins from the soil bacteria Streptomyces having over 250 ESTs in a total of ∼2,000 ESTs, suggesting the clavuligerus, having 52% identity to the insect proteins. OBP family has been recruited by Cimex to function as the Similarly, the protein originally described in R. prolixus lipocalins in triatomines. No salivary member of this family as MYS2 has homologs found in the sialotranscriptomes in Cimicomorpha has been so far functionally characterized. of T. brasiliensis and T. matogrossensis and is similar to many other insect proteins in the NR, including protein sequences deducted from the sialotranscriptome of the tsetse 3.5. Antigen-5 Family. This is a ubiquitous protein family Glossina morsitans [166]. Three sequences, one each from found in plants and animals, including expression in the C. lectularius, T. infestans, and T. matogrossensis, have 25% venom glands of vespids, where it was recognized as an amino acid sequence identity but 52% similarity and little antigen, thus the name antigen 5 for this family. They are similarity to other proteins on the NR database. These members of the CAP superfamily, most with unknown func- sequences are grouped in Additional File 1 as the Cimex- tion [157]. In snakes and lizards, they have been associated Triatoma family. PSI-blast initiated by the T. matogrossensis with venom toxins [158–160]. In stable flies, one salivary sequence against the NR database initially retrieves only antigen 5 protein binds immunoglobulins and may function the two other sequences, but on first iteration it retrieves as an inhibitor of the classical pathway of complement dozens of insect proteins (Additional File 3), and in the third activation [161]. In horse flies, one protein has acquired iteration it retrieves Daphnia and tick proteins, suggesting a disintegrin motif and is a strong inhibitor of platelet this is an arthropod family of high divergence. Finally, aggregation [162–164]. All triatomine sialotranscriptomes the sialotranscriptome of T. matogrossensis identified four have revealed this class of proteins, which is particularly additional nonrelated proteins that have insect homologs abundant in Dipetalogaster. The function of these proteins but were not found in other reported sialotranscriptomes in triatomine blood feeding is still unknown. of Hemiptera but are similar to proteins reported from G. morsitans and from Aedes aegypti sialotranscriptomes. It 3.6. Lectin. Triatoma dimidiata exclusively presents two par- is possible that these families function as antimicrobial tial sequences containing a galactose-binding domain. While peptides, but so far none has been characterized. lectins—mainly C-type lectins—have been described in the sialotranscriptome of mosquitoes (none with known 5. Hemiptera-Specific Families function), this is so far a unique finding in triatomine sialo- transcriptomes. 5.1. Mys3/Hemolysin Family. When the R. prolixus sialotran- scriptome was reported [36], an additional mysterious pro- 3.7. Immunity-Related, Ubiquitous Families. Immunity- tein was named Mys3. Later, with additional sialotranscrip- related proteins and peptides are commonly found in the tome reports, another protein family emerged, named as saliva of bloodsucking arthropods and may help to control hemolysin-like because some members had weak similarity microbial growth in the ingested meal and perhaps also to bacterial proteins annotated as hemolysins. PSI-blast later avoid microbial infection of the bite site. Lysozyme, while revealed that these proteins all belong to a single family that common in mosquito sialomes, is found exclusively so far is quite divergent, including a truncated protein from the in Cimex sialomes, with four quite different proteins being sialome of Oncopeltus, suggesting a non-blood-feeding role, reported. D. maxima presents a histidine-rich peptide that perhaps antimicrobial, for its members. 10 Psyche
5.2. Triatoma-Specific Families. Sialotranscriptomes of sev- 7. Concluding Remarks eral species of the Triatoma genusrevealseveralunique protein families, among which are the trialysin and short Blood-feeding Cimicomorpha have developed a sophisti- trialysin families. The trialysins are basic proteins of mature cated and divergent array of salivary pharmacologically MW near 26 kDa that can be further processed to peptides active compounds that disarm their hosts’ reaction against that have lytic properties [17, 18] and may function as blood loss. In a few transcriptomes encompassing members antimicrobials. Short trialysins have mature MW of ∼6.1 of the Reduviidae and Cimicidae, the convergent evolution scenario in the sialomes of these two families is apparent. and acidic pI and are so named because they match the ff amino terminal region of the mature trialysins. Both forms Both have apyrase activity, but from di erent gene families; Cimex and Rhodnius (but not any Triatomini member) are abundantly expressed but only found in T. infestans and use NO as a vasodilator but co-opted completely different T. matogrossensis, which are from southern South America, heme proteins to carry this unstable gas. The anticlotting and are not found in the sialotranscriptomes of T. brasiliensis, compounds are different at the Reduviidae tribe level and found in northeastern Brazil, or on those of the North so on. The lipocalin expansion is remarkable among the American T. dimidiata or T. rubida. Additional File 1 reports triatomines and nonexistent in Cimex. These proteins can 19 protein sequences from Triatoma that are not similar to play many different functions as binders of small agonists anything deposited in the NR database and two pairs of (kratagonists), NO carriers, or protease inhibitors. In Cimex, sequences from T. matogrossensis that only match its pair the expanded odorant binding family may have taken this members. None has been functionally characterized. It is role, but none thus far has been characterized. interesting that of these 23 sequences only one derives from Notice that the sialome of Oncopeltus, a member of the T. rubida and the remaining derive from T. infestans and T. Pentatomomorpha—the most closely related suborder to the matogrossensis, although the number of clones sequenced for Cimicomorpha (see http://tolweb.org/Heteroptera/10805) the T. brasiliensis, T. dimidiata, and T. rubida was similar [76, 167]—revealed virtually nothing in common with the to those of T. infestans and T. matogrossensis, suggesting a Cimicomorpha, and the Cimicidae sialome also revealed greater sialome diversity in these bugs from southern South little in common with the Reduviidae, perhaps as expected by America. the divergence of these families (see http://tolweb.org/Cim- icomorpha/10817). Zooming-in on the Triatomine group, it 5.3. Rhodnius-, Cimex-, and Oncopeltus-Specific Families. will be interesting in the future to describe the sialomes of Additional File 1 presents 16 proteins from the bugs named additional tribes of the Triatomine, such as the Bolboderini, above that have no significant matches to the NR database which includes bugs that feed on insect hemolymph, the except in some cases for some proteins of low complexity. Cavernicolini that are associated with bats, and members None of these proteins has been functionally characterized. of the Linshcosteus genus that are found in India [168] This includes Rhodnius MY1 protein, one of three mysterious and could be divergent members. Zooming a little out proteins revealed in the first bug sialotranscriptome [36]. As and as indicated by Schofield and Galvao˜ [49], facultative seen above, MYS2 and MYS3 were later found to be members blood feeding is found in non-Triatominae members of of larger families. It is expected that, with a larger number the Reduviidae, including the Emesinae, Harpactorinae, of genomes and transcriptomes sequenced, MYS1—as well Peiratinae, Physoderinae, and Reduviinae. Sialomes of these as the other orphan proteins in this group—will also be subfamilies could be more indicative of the prevalent “pre- deorphanized. adaptations” available as stepping stones and promoted by the blood-feeding habit. On the other hand, the Cimi- cidae are closely related to the bat bugs (Polyctenidae), 6. Housekeeping Proteins which is a sister group, and to the Anthocoridae (flower bugs; http://tolweb.org/Cimicomorpha/10817), which feed Mostly from the sialotranscriptomes shown in Table 2, on small insects. These non-blood-feeding closer relatives many housekeeping protein sequences were also deduced, may reveal insights into the Cimicidae evolution to hema- including many associated with energy metabolism, protein tophagy. synthesis, modification, and export, among other classes (see worksheet named “Housekeeping” of Additional File 1). Acknowledgments Interestingly, the sialotranscriptome of Triatoma rubida shows abundant expression of members of the cytochrome This work was supported by the Intramural Research Pro- P450 as well as of the 15-hydroxyprostaglandin dehydroge- gram of the Division of Intramural Research, the National nase, suggesting either that the salivary gland may have an Institute of Allergy and Infectious Diseases, and the National active endogenous prostaglandin signaling or that prostagl- Institutes of Health. The authors thank NIAID DIR intramu- andins may be secreted in the saliva of these bugs. Cyt P450 ral editor Brenda Rae Marshall for editing the manuscript. transcripts were also detected in Rhodnius and T. mato- Because JMCR and IMBF are government employees and grossensis, and the prostaglandin dehydrogenase was also this is a government work, the work is in the public found in T. infestans. Increased depth of sequencing of these domain in the United States. Notwithstanding any other sialotranscriptomes may certainly reveal these two classes of agreements, the NIH reserves the right to provide the work proteins to be expressed in all triatomines. to PubMedCentral for display and use by the public, and Psyche 11
PubMedCentral may tag or modify the work consistent with [13]E.Faudry,J.M.Santana,C.Ebel,T.Vernet,andA.R.L.Teix- its customary practices. You can establish rights outside of eira, “Salivary apyrases of Triatoma infestans are assembled the US subject to a government use license. into homo-oligomers,” Biochemical Journal, vol. 396, no. 3, pp. 509–515, 2006. [14] A. Morita, H. Isawa, Y. Orito, S. Iwanaga, Y. Chinzei, and References M. Yuda, “Identification and characterization of a collagen- induced platelet aggregation inhibitor, triplatin, from sali- [1] D. Grimaldi and M. Engel, Evolution of the Insects, Cambridge vary glands of the assassin bug, Triatoma infestans,” FEBS University Press, New York, NY, USA, 2005. Journal, vol. 273, no. 13, pp. 2955–2962, 2006. [2] J. M. C. Ribeiro, M. Schneider, and J. A. Guimaraes, “Purifi- [15] D. Ma, T. C. F. Assumpcao, Y. Li, J. F. Andersen, J. Ribeiro, cation and characterization of prolixin S (nitrophorin 2), I. M. B. Francischetti et al., “Triplatin, a platelet aggregation the salivary anticoagulant of the blood-sucking bug Rhodnius inhibitor from the salivary gland of the triatomine vector prolixus,” Biochemical Journal, vol. 308, no. 1, pp. 243–249, of chagas disease, binds to TXA2 but does notinteract with 1995. GPVI,” Thrombosis and Haemostasis, vol. 1, no. 107, pp. 111– [3]I.M.B.Francischetti,J.F.Andersen,andJ.M.C.Ribeiro, 123, 2011. “Biochemical and functional characterization of recombi- [16] H. Isawa, Y. Orito, N. Jingushi et al., “Identification and nant Rhodnius prolixus platelet aggregation inhibitor 1 as a ffi characterization of plasma kallikrein-kinin system inhibitors novel lipocalin with high a nity for adenosine diphosphate from salivary glands of the blood-sucking insect Triatoma and other adenine nucleotides,” Biochemistry, vol. 41, no. 11, infestans,” FEBS Journal, vol. 274, no. 16, pp. 4271–4286, pp. 3810–3818, 2002. 2007. [4] I. M. B. Francischetti, J. M. C. Ribeiro, D. Champagne, [17] R. M. Martins, M. L. Sforc¸a, R. Amino et al., “Lytic activity and J. Andersen, “Purification, cloning, expression, and and structural differences of amphipathic peptides derived mechanism of action of a novel platelet aggregation inhibitor from trialysin,” Biochemistry, vol. 45, no. 6, pp. 1765–1774, from the salivary gland of the blood-sucking bug, Rhodnius 2006. prolixus,” The Journal of Biological Chemistry, vol. 275, no. 17, [18]R.Amino,R.M.Martins,J.Procopio,I.Y.Hirata,M.A. pp. 12639–12650, 2000. Juliano, and S. Schenkman, “Trialysin, a novel pore-forming [5] J. F. Andersen and W. R. Montfort, “The crystal structure of protein from saliva of hematophagous insects activated by nitrophorin 2. A trifunctional antihemostatic protein from limited proteolysis,” The Journal of Biological Chemistry, vol. the saliva of Rhodnius prolixus,” The Journal of Biological 277, no. 8, pp. 6207–6213, 2002. Chemistry, vol. 275, no. 39, pp. 30496–30503, 2000. [19] C. Noeske-Jungblut, J. Kratzschmar,¨ B. Haendler et al., “An ffi [6]J.M.C.RibeiroandF.A.Walker,“Higha nity histamine- inhibitor of collagen-induced platelet aggregation from the binding and antihistaminic activity of the salivary nitric saliva of Triatoma pallidipennis,” The Journal of Biological oxide-carrying heme protein (nitrophorin) of Rhodnius Chemistry, vol. 269, no. 7, pp. 5050–5053, 1994. prolixus,” Journal of Experimental Medicine, vol. 180, no. 6, [20] B. Haendler, A. Becker, C. Noeske-Jungblut, J. Kratzschmar, pp. 2251–2257, 1994. P. Donner, and W. D. Schleuning, “Expression of active re- [7] J. F. Andersen, I. M. B. Francischetti, J. G. Valenzuela, P. combinant pallidipin, a novel platelet aggregation inhibitor, Schuck, and J. M. C. Ribeiro, “Inhibition of hemostasis by in the periplasm of Escherichia coli,” Biochemical Journal, vol. a high affinity biogenic amine-binding protein from the 307, no. 2, pp. 465–470, 1995. saliva of a blood-feeding insect,” The Journal of Biological [21] C. Noeske-Jungblut, B. Haendler, P. Donner, A. Alagon, L. Chemistry, vol. 278, no. 7, pp. 4611–4617, 2003. Possani, and W. D. Schleuning, “Triabin, a highly potent [8] J. F. Andersen and J. M. C. Ribeiro, “A secreted salivary exosite inhibitor of thrombin,” The Journal of Biological inositol polyphosphate 5-phosphatase from a blood-feeding Chemistry, vol. 270, no. 48, pp. 28629–28634, 1995. insect: allosteric activation by soluble phosphoinositides and [22] E. Glusa, E. Bretschneider, J. Daum, and C. Noeske-Jungblut, phosphatidylserine,” Biochemistry, vol. 45, no. 17, pp. 5450– “Inhibition of thrombin-mediated cellular effects by triabin, 5457, 2006. a highly potent anion-binding exosite thrombin inhibitor,” [9]D.M.Golodne,R.Q.Monteiro,A.V.Grac¸a-Souza,M.A.C. Thrombosis and Haemostasis, vol. 77, no. 6, pp. 1196–1200, Silva-Neto, and G. C. Atella, “Lysophosphatidylcholine acts 1997. as an anti-hemostatic molecule in the saliva of the blood- [23] P. Fuentes-Prior, C. Noeske-Jungblut, P. Donner, W. D. sucking bug Rhodnius prolixus,” The Journal of Biological Schleuning, R. Huber, and W. Bode, “Structure of the throm- Chemistry, vol. 278, no. 30, pp. 27766–27771, 2003. bin complex with triabin, a lipocalin-like exosite-binding [10] J. M. C Ribeiro, J. M. H Hazzard, R. H. Nussenzveig, D. inhibitor derived from a triatomine bug,” Proceedings of the E. Champagne, F. A. Walker et al., “Reversible binding of National Academy of Sciences of the United States of America, nitric oxide by a salivary nitrosylhemeprotein from the blood vol. 94, no. 22, pp. 11845–11850, 1997. sucking bug, Rhodnius prolixus,” Science, vol. 260, Article ID [24]C.D.Paddock,J.H.McKerrow,E.Hansell,K.W.Foreman,I. 5107, pp. 539–541, 1993. Hsieh, and N. Marshall, “Identification, cloning, and recom- [11] D. E. Champagne, R. H. Nussenzveig, and J. M. C. Ribeiro, binant expression of procalin, a major triatomine allergen,” “Purification, partial characterization, and cloning of nitric Journal of Immunology, vol. 167, no. 5, pp. 2694–2699, 2001. oxide- carrying heme proteins (nitrophorins) from salivary [25] T. C. F. Assumpc¸ao,P.H.Alvarenga,J.M.C.Ribeiro,J.˜ glands of the blood-sucking insect Rhodnius prolixus,” The F. Andersen, and I. M. B. Francischetti, “Dipetalodipin, a Journal of Biological Chemistry, vol. 270, no. 15, pp. 8691– novel multifunctional salivary lipocalin that inhibits platelet 8695, 1995. aggregation, vasoconstriction, and angiogenesis through [12] E. Faudry, S. P. Lozzi, J. M. Santana et al., “Triatoma infestans unique binding specificity: TXA2, PGF2α, and 15(S)-HETE,” apyrases belong to the 5 -nucleotidase family,” The Journal of The Journal of Biological Chemistry, vol. 285, no. 50, pp. Biological Chemistry, vol. 279, no. 19, pp. 19607–19613, 2004. 39001–39012, 2010. 12 Psyche
[26] J. G. Valenzuela, R. Charlab, M. Y. Galperin, and J. M. C. (Hemiptera, Triatominae),” Insect Biochemistry and Molecu- Ribeiro, “Purification, cloning, and expression of an apyrase lar Biology, vol. 37, no. 7, pp. 702–712, 2007. from the bed bug Cimex lectularius: a new type of nucleotide- [40] T. C. Assumpc¸ao,˜ D. P. Eaton, V. M. Pham et al., “An insight binding enzyme,” The Journal of Biological Chemistry, vol. into the sialotranscriptome of Triatoma matogrossensis, a 273, no. 46, pp. 30583–30590, 1998. kissing bug associated with fogo selvagem in South America,” [27] J. G. Valenzuela, F. A. Walker, and J. M. Ribeiro, “A salivary American Journal of Tropical Medicine and Hygiene, vol. 86, nitrophorin (nitric-oxide-carrying hemoprotein) in the bed- no. 6, pp. 1005–1014, 2012. bug Cimex lectularius,” Journal of Experimental Biology, vol. [41] J. M. Ribeiro, T. C. Assumpc¸ao,˜ V. M. Pham, I. M. Francis- 198, pp. 1519–1526, 1995. chetti, C. E. Reisenman et al., “An insight into the sialotran- [28] J. G. Valenzuela and J. M. C. Ribeiro, “Purification and scriptome of Triatoma rubida (Hemiptera: Heteroptera),” cloning of the salivary nitrophorin from the hemipteran Journal of Medical Entomology, vol. 49, no. 3, pp. 563–572, Cimex lectularius,” Journal of Experimental Biology, vol. 201, 2012. no. 18, pp. 2659–2664, 1998. [42]H.Kato,R.C.Jochim,E.A.Gomezetal.,“Arepertoireofthe [29] A. Weichsel, E. M. Maes, J. F. Andersen et al., “Heme-assisted dominant transcripts from the salivary glands of the blood- S-nitrosation of a proximal thiolate in a nitric oxide transport sucking bug, Triatoma dimidiata, a vector of Chagas disease,” protein,” Proceedings of the National Academy of Sciences of the Infection, Genetics and Evolution, vol. 10, no. 2, pp. 184–191, United States of America, vol. 102, no. 3, pp. 594–599, 2005. 2010. [30] C. K. Meiser, H. Piechura, H. E. Meyer, B. Warscheid, G. A. [43] T. C. F. Assumpc¸ao,˜ S. Charneau, P. B. M. Santiago et al., Schaub, and C. Balczun, “A salivary serine protease of the “Insight into the salivary transcriptome and proteome of haematophagous reduviid Panstrongylus megistus: sequence Dipetalogaster maxima,” Journal of Proteome Research, vol. 10, characterization, expression pattern and characterization of no. 2, pp. 669–679, 2011. proteolytic activity,” Insect Molecular Biology, vol. 19, no. 3, [44] A. C. M. Bussacos, E. S. Nakayasu, M. M. Hecht et al., pp. 409–421, 2010. “Redundancy of proteins in the salivary glands of Panstrongy- [31] W. G. Friend and J. J. B. Smith, “Feeding in Rhodnius prolixus: lus megistus secures prolonged procurement for blood mouthpart activity and salivation, and their correlation with meals,” Journal of Proteomics, vol. 74, no. 9, pp. 1693–1700, changes of electrical resistance,” Journal of Insect Physiology, 2011. vol. 17, no. 2, pp. 233–243, 1971. [45] I. M. B. Francischetti, E. Calvo, J. F. Andersen et al., “Insight [32] A. C. Soares, J. Carvalho-Tavares, N. D. F. Gontijo, V. C. dos into the sialome of the bed bug, Cimex lectularius,” Journal of Santos, M. M. Teixeira, and M. H. Pereira, “Salivation pattern Proteome Research, vol. 9, no. 8, pp. 3820–3831, 2010. of Rhodnius prolixus (Reduviidae; Triatominae) in mouse [46] I. M. B. Francischetti, A. H. Lopes, F. A. Dias, V. M. Pham, skin,” JournalofInsectPhysiology, vol. 52, no. 5, pp. 468–472, and J. M. C. Ribeiro, “An insight into the sialotranscriptome 2006. of the seed-feeding bug, oncopeltus fasciatus,” Insect Bio- [33] M. M. Lavoipierre, G. Dickerson, and R. M. Gordon, “Studies chemistry and Molecular Biology, vol. 37, no. 9, pp. 903–910, on the methods of feeding of blood-sucking arthropods. I. 2007. The manner in which triatomine bugs obtain their blood- [47] R. H. Cobben et al., “On the original feeding habits of the meal, as observed in the tissues of the living rodent, with hemiptera (Insecta): a reply to Merrill Sweet,” Annals of the some remarks on the effects of the bite on human volunteers,” Entomological Society of America, vol. 72, no. 6, pp. 711–715, Annals of Tropical Medicine and Parasitology, vol. 53, pp. 235– 1979. 250, 1959. [48] V. Hypsa,D.F.Tietz,J.Zrzavˇ y,´ R. O. M. Rego, C. Galvao, [34] S. Dinant, J. L. Bonnemain, C. Girousse, and J. Kehr, “Phloem and J. Jurberg, “Phylogeny and biogeography of triatominae sap intricacy and interplay with aphid feeding,” Comptes (Hemiptera: Reduviidae): molecular evidence of a New world Rendus, vol. 333, no. 6-7, pp. 504–515, 2010. origin of the asiatic clade,” Molecular Phylogenetics and [35] J. M. C. Ribeiro et al., “Insect saliva: function, biochemistry Evolution, vol. 23, no. 3, pp. 447–457, 2002. and physiology,” in Regulatory Mechanisms of Insect Feeding, [49] C. J. Schofield and C. Galvao,˜ “Classification, evolution, and R. F. Chapman and G. de Boer, Eds., pp. 74–97, Chapman & species groups within the triatominae,” Acta Tropica, vol. 110, Hall, London, 1995. no. 2-3, pp. 88–100, 2009. [36] J. M. C. Ribeiro, J. Andersen, M. A. C. Silva-Neto, V. M. [50] J. G. Valenzuela, O. M. Chuffe,andJ.M.C.Ribeiro,“Apyrase Pham, M. K. Garfield, and J. G. Valenzuela, “Exploring the and anti-platelet activities from the salivary glands of the sialome of the blood-sucking bug Rhodnius prolixus,” Insect bed bug Cimex lectularius,” Insect Biochemistry and Molecular Biochemistry and Molecular Biology, vol. 34, no. 1, pp. 61–79, Biology, vol. 26, no. 6, pp. 557–562, 1996. 2004. [51]E.Faudry,P.S.Rocha,T.Vernet,S.P.Lozzi,andA.R. [37] A. C. M. Bussacos, E. S. Nakayasu, M. M. Hecht et al., L. Teixeira, “Kinetics of expression of the salivary apyrases “Diversity of anti-haemostatic proteins in the salivary glands in Triatoma infestans,” Insect Biochemistry and Molecular of Rhodnius species transmitters of Chagas disease in the Biology, vol. 34, no. 10, pp. 1051–1058, 2004. greater Amazon,” Journal of Proteomics,vol.74,no.9,pp. [52] W. R. Montfort, A. Weichsel, and J. F. Andersen, “Nitr- 1664–1672, 2011. ophorins and related antihemostatic lipocalins from Rhod- [38] T. C. F. Assumpc¸ao,I.M.B.Francischetti,J.F.Andersen,A.˜ nius prolixus and other blood-sucking arthropods,” Biochim- Schwarz, J. M. Santana, and J. M. C. Ribeiro, “An insight into ica et Biophysica Acta, vol. 1482, no. 1-2, pp. 110–118, 2000. the sialome of the blood-sucking bug Triatoma infestans,a [53] J. F. Andersen, D. E. Champagne, A. Weichsel et al., “Nitric vector of Chagas’ disease,” Insect Biochemistry and Molecular oxide binding and crystallization of recombinant nitrophorin Biology, vol. 38, no. 2, pp. 213–232, 2008. I, a nitric oxide transport protein from the blood-sucking bug [39]A.Santos,J.M.C.Ribeiro,M.J.Lehaneetal.,“Thesialo- Rhodnius prolixus,” Biochemistry, vol. 36, no. 15, pp. 4423– transcriptome of the blood-sucking bug Triatoma brasiliensis 4428, 1997. Psyche 13
[54] A. Weichsel, J. F. Andersen, D. E. Champagne, F. A. Walker, [71] D. L. Wheeler, T. Barrett, D. A. Benson et al., “Database and W. R. Montfort, “Crystal structures of a nitric oxide resources of the National Center for Biotechnology Informa- transport protein from a blood-sucking insect,” Nature tion,” Nucleic Acids Research, vol. 33, pp. D39–D45, 2005. Structural Biology, vol. 5, no. 4, pp. 304–309, 1998. [72]A.Marchler-Bauer,A.R.Panchenko,B.A.Shoemarker,P.A. [55] J. F. Andersen, A. Weichsel, C. A. Balfour, D. E. Champagne, Thiessen, L. Y. Geer, and S. H. Bryant, “CDD: a database of and W. R. Montfort, “The crystal structure of nitrophorin 4 conserved domain alignments with links to domain three- at 1.5 A˚ resolution: transport of nitric oxide by a lipocalin- dimensional structure,” Nucleic Acids Research, vol. 30, no. 1, based heme protein,” Structure, vol. 6, no. 10, pp. 1315–1327, pp. 281–283, 2002. 1998. [73] H. Nielsen, S. Brunak, and G. von Heijne, “Machine learning [56] I. M. B. Francischetti, A. Sa-Nunes,´ B. J. Mans, I. M. Santos, approaches for the prediction of signal peptides and other and J. M. C. Ribeiro, “The role of saliva in tick feeding,” protein sorting signals,” Protein Engineering,vol.12,no.1, Frontiers in Bioscience, vol. 14, no. 6, pp. 2051–2088, 2009. pp. 3–9, 1999. [57] I. M. B. Francischetti, “Platelet aggregation inhibitors from [74] E. L. Sonnhammer, G. von Heijne, and A. Krogh, “A hidden hematophagous animals,” Toxicon, vol. 56, no. 7, pp. 1130– Markov model for predicting transmembrane helices in 1144, 2010. protein sequences,” Proceedings International Conference on [58]J.W.CornwallandW.S.Patton,“Someobservationsonthe Intelligent Systems for Molecular Biology, vol. 6, pp. 175–182, salivary secretion of the common blood-sucking insects and 1998. ticks,” Indian Journal of Medical Research, vol. 2, pp. 569–593, [75] J. E. Hansen, O. Lund, N. Tolstrup, A. A. Gooley, K. L. 1914. Williams, and S. Brunak, “NetOglyc: prediction of mucin [59] J. M. C. Ribeiro and E. S. Garcia, “Platelet antiaggregating type O-glycosylation sites based on sequence context and activity in the salivary secretion of the blood sucking bug surface accessibility,” Glycoconjugate Journal, vol. 15, no. 2, Rhodnius prolixus,” Experientia, vol. 37, no. 4, pp. 384–386, pp. 115–130, 1998. 1981. [76] “Heteroptera: true bugs,” http://tolweb.org/Heteroptera/ [60] M. J. Mant and K. R. Parker, “Two platelet aggregation 10805/2009.02.27. inhibitors in tsetse (Glossina) saliva with studies of roles of [77] H. M. Kalckar, “Adenylpyrophosphatase and myokinase,” The Journal of Biological Chemistry, vol. 153, pp. 355–373, 1945. thrombine and citrate in in vitro platelet aggregation,” British ff Journal of Haematology, vol. 48, no. 4, pp. 601–608, 1981. [78] O. Meyerho et al., “The origin of the reaction of Harden and Young in cell-free alcoholic fermentation,” The Journal [61]J.M.C.Ribeiro,A.Vachereau,G.B.Modi,andR.B.Tesh, of Biological Chemistry, vol. 157, pp. 105–119, 1945. “A novel vasodilatory peptide from the salivary glands of the [79] P. S. Krishnam, “Studies on apyrase. II: some properties of sand fly Lutzomyia longipalpis,” Science, vol. 243, no. 4888, potato apyrase,” Archives of Biochemistry,vol.20,no.2,pp. pp. 212–214, 1989. 272–283, 1949. [62] K. Hellmann and R. I. Hawkins, “Prolixin-S and Prolixin-G; [80] K. H. Lee, J. Z. Ksezanoski, J. J. Eiler et al., “Mode of action two anticoagulants from Rhodnius prolixus Stal,”˚ Nature, vol. of potato apyrase,” Proceedings of the Society for Experimental 207, no. 4994, pp. 265–267, 1965. Biology and Medicine, vol. 1, no. 94, pp. 193–195, 1957. [63] J. J. B. Smith, R. A. Cornish, and J. Wilkes, “Properties of a [81] X. D. Gao, V. Kaigorodov, and Y. Jigami, “YND1, a calcium-dependent apyrase in the saliva of the blood-feeding homologue of GDA1, encodes membrane-bound apyrase bug, Rhodnius prolixus,” Experientia, vol. 36, no. 8, pp. 898– required for Golgi N- and O-glycosylation in Saccharomyces 900, 1980. cerevisiae,” The Journal of Biological Chemistry, vol. 274, no. [64] J. M. C. Ribeiro, R. Gonzales, and O. Marinotti, “A salivary 30, pp. 21450–21456, 1999. vasodilator in the blood-sucking bug, Rhodnius prolixus,” [82] C. D’Alessio, E. S. Trombetta, and A. J. Parodi, “Nucleoside British Journal of Pharmacology, vol. 101, no. 4, pp. 932–936, diphosphatase and glycosyltransferase activities can local- 1990. ize to different subcellular compartments in Schizosaccha- [65] R. R. Cavalcante, M. H. Pereira, and N. F. Gontijo, “Anti- romyces pombe,” The Journal of Biological Chemistry, vol. complement activity in the saliva of phlebotomine sand flies 278, no. 25, pp. 22379–22387, 2003. and other haematophagous insects,” Parasitology, vol. 127, [83] J. J. Sarkis, J. A. Guimaraes,˜ and J. M. Ribeiro, “Salivary no. 1, pp. 87–93, 2003. apyrase of Rhodnius prolixus. Kinetics and purification,” [66] J. M. C. Ribeiro, “The antiserotonin and antihistamine Biochemical Journal, vol. 233, no. 3, pp. 885–891, 1986. activities of salivary secretion of Rhodnius prolixus,” Journal [84] J. M. C. Ribeiro and E. S. Garcia, “The salivary and crop of Insect Physiology, vol. 28, no. 1, pp. 69–75, 1982. apyrase activity of Rhodnius prolixus,” Journal of Insect [67] J. M. C. Ribeiro and J. J. F. Sarkis, “Anti-thromboxane activity Physiology, vol. 26, no. 5, pp. 303–307, 1980. in Rhodnius prolixus salivary secretion,” Journal of Insect [85] J. M. C. Ribeiro, J. J. F. Sarkis, P. A. Rossignol, and A. Physiology, vol. 28, no. 8, pp. 655–660, 1982. Spielman, “Salivary apyrase of Aedes aegypti: characterization [68] A.´ Dan, M. H. Pereira, J. L. Pesquero, L. Diotaiuti, and P. and secretory fate,” Comparative Biochemistry and Physiology S. Lacerda Beirao,˜ “Action of the saliva of Triatoma infestans B, vol. 79, no. 1, pp. 81–86, 1984. (Heteroptera: Reduviidae) on sodium channels,” Journal of [86] J. M. C. Ribeiro, P. A. Rossignol, and A. Spielman, “Sali- Medical Entomology, vol. 36, no. 6, pp. 875–879, 1999. vary gland apyrase determines probing time in anopheline [69] S. F. Altschul, T. L. Madden, A. A. Scha¨ffer et al., “Gapped mosquitoes,” Journal of Insect Physiology, vol. 31, no. 9, pp. BLAST and PSI-BLAST: a new generation of protein database 689–692, 1985. search programs,” Nucleic Acids Research, vol. 25, no. 17, pp. [87] D. E. Champagne, C. T. Smartt, J. M. C. Ribeiro, and A. A. 3389–3402, 1997. James, “The salivary gland-specific apyrase of the mosquito [70] M. Ashburner, C. A. Ball, J. A. Blake et al., “Gene ontology: Aedes aegypti is a member of the 5’-nucleotidase family,” tool for the unification of biology,” Nature Genetics, vol. 25, Proceedings of the National Academy of Sciences of the United no. 1, pp. 25–29, 2000. States of America, vol. 92, no. 3, pp. 694–698, 1995. 14 Psyche
[88] J. Schulte Am Esch, J. Sevigny,´ E. Kaczmarek et al., “Structural deaminase from the sand fly Phlebotomus duboscqi, the elements and limited proteolysis of CD39 influence ATP vector of Leishmania major in sub-Saharan Africa,” Journal diphosphohydrolase activity,” Biochemistry,vol.38,no.8,pp. of Experimental Biology, vol. 210, no. 5, pp. 733–740, 2007. 2248–2258, 1999. [105] S. J. Harris, R. V. Parry, J. Westwick, and S. G. Ward, [89] K. T. Tan, S. P. Watson, and G. Y. H. Lip, “The endothelium “Phosphoinositide lipid phosphatases: natural regulators of and platelets in cardiovascular disease: potential targets for phosphoinositide 3-kinase signaling in T lymphocytes,” The therapeutic intervention,” Current Medicinal Chemistry, vol. Journal of Biological Chemistry, vol. 283, no. 5, pp. 2465– 2, no. 2, pp. 169–178, 2004. 2469, 2008. [90] A. J. Marcus, M. J. Broekman, J. H. F. Drosopoulos et al., [106] S. P. Watson, B. Reep, R. T. McConnell, and E. G. Lapetina, “Role of CD39 (NTPDase-1) in thromboregulation, cerebro- “Collagen stimulates [3H]inositol triphosphate formation in protection, and cardioprotection,” Seminars in Thrombosis indomethacin-treated human platelets,” Biochemical Journal, and Hemostasis, vol. 31, no. 2, pp. 234–246, 2005. vol. 226, no. 3, pp. 831–837, 1985. [91] B. U. Failer, N. Braun, and H. Zimmermann, “Cloning, [107] R. Amino, A. S. Tanaka, and S. Schenkman, “Triapsin, an expression, and functional characterization of a Ca2+- unusual activatable serine protease from the saliva of the dependent endoplasmic reticulum nucleoside diphospha- hematophagous vector of Chagas’ disease Triatoma infestans tase,” The Journal of Biological Chemistry, vol. 277, no. 40, pp. (Hemiptera: Reduviidae),” Insect Biochemistry and Molecular 36978–36986, 2002. Biology, vol. 31, no. 4-5, pp. 465–472, 2001. [92]C.Devader,R.J.Webb,G.M.H.Thomas,andL.Dale, [108] G. Colebatch, P. Cooper, and P. East, “cDNA cloning of a sali- “Xenopus apyrase (xapy), a secreted nucleotidase that is vary chymotrypsin-like protease and the identification of six expressed during early development,” Gene, vol. 367, no. 1- additional cDNAs encoding putative digestive proteases from 2, pp. 135–141, 2006. the green mirid, Creontiades dilutus (Hemiptera: Miridae),” [93] J. G. Valenzuela, Y. Belkaid, E. Rowton, and J. M. C. Ribeiro, Insect Biochemistry and Molecular Biology,vol.32,no.9,pp. “The salivary apyrase of the blood-sucking sand fly Phleboto- 1065–1075, 2002. mus papatasi belongs to the novel Cimex family of apyrases,” [109] Y. C. Zhu, F. Zeng, and B. Oppert, “Molecular cloning of Journal of Experimental Biology, vol. 204, no. 2, pp. 229–237, trypsin-like cDNAs and comparison of proteinase activities 2001. in the salivary glands and gut of the tarnished plant bug [94] D. Sun, A. Mcnicol, A. A. James, and Z. Peng, “Expression Lygus lineolaris (Heteroptera: Miridae),” Insect Biochemistry of functional recombinant mosquito salivary apyrase: a and Molecular Biology, vol. 33, no. 9, pp. 889–899, 2003. potential therapeutic platelet aggregation inhibitor,” Platelets, [110] F. Zeng, Y. C. Zhu, and A. C. Cohen, “Molecular cloning vol. 17, no. 3, pp. 178–184, 2006. and partial characterization of a trypsin-like protein in [95] M. Andersson, “Diadenosine tetraphosphate (Ap4A): its salivary glands of Lygus hesperus (hemiptera: Miridae),” presence and functions in biological systems,” International Insect Biochemistry and Molecular Biology,vol.32,no.4,pp. Journal of Biochemistry, vol. 21, no. 7, pp. 707–714, 1989. 455–464, 2002. [96] H. Schluter,¨ M. Tepel, and W. Zidek, “Vascular actions of [111] I. M. B. Francischetti, T. N. Mather, and J. M. C. Ribeiro, diadenosine phosphates,” Journal of Autonomic Pharmacol- “Cloning of a salivary gland metalloprotease and character- ogy, vol. 16, no. 6, pp. 357–362, 1996. ization of gelatinase and fibrin(ogen)lytic activities in the [97]L.L.Kisselev,J.Justesen,A.D.Wolfson,andL.Y.Frolova, saliva of the Lyme disease tick vector Ixodes scapularis,” “Diadenosine oligophosphates (AP(n)A), a novel class of Biochemical and Biophysical Research Communications, vol. signalling molecules?” FEBS Letters, vol. 427, no. 2, pp. 157– 305, no. 4, pp. 869–875, 2003. 163, 1998. [112] I. M. B. Francischetti, T. N. Mather, and J. M. C. Ribeiro, [98] B. M. Stavrou, “Diadenosine polyphosphates: postulated “Tick saliva is a potent inhibitor of endothelial cell prolifera- mechanisms mediating the cardiac effects,” Current Medici- tion and angiogenesis,” Thrombosis and Haemostasis, vol. 94, nal Chemistry, vol. 1, no. 2, pp. 151–169, 2003. no. 1, pp. 167–174, 2005. [99] E. Calvo and J. M. C. Ribeiro, “A novel secreted endonuclease [113] M. P. Williamson, D. Marion, and K. Wuthrich,¨ “Secondary from Culex quinquefasciatus salivary glands,” Journal of structure in the solution conformation of the proteinase Experimental Biology, vol. 209, no. 14, pp. 2651–2659, 2006. inhibitor IIA from bull seminal plasma by nuclear magnetic [100] J. G. Valenzuela, M. Garfield, E. D. Rowton, and V. M. Pham, resonance,” Journal of Molecular Biology, vol. 173, no. 3, pp. “Identification of the most abundant secreted proteins from 341–359, 1984. the salivary glands of the sand fly Lutzomyia longipalpis,vec- [114] I. T. N. Campos, R. Amino, C. A. M. Sampaio et al., tor of Leishmania chagasi,” Journal of Experimental Biology, “Infestin, a thrombin inhibitor presents in Triatoma infestans vol. 207, no. 21, pp. 3717–3729, 2004. midgut, a Chagas’ disease vector: gene cloning, expression [101] J. M. Anderson, F. Oliveira, S. Kamhawi et al., “Comparative and characterization of the inhibitor,” Insect Biochemistry and salivary gland transcriptomics of sandfly vectors of visceral Molecular Biology, vol. 32, no. 9, pp. 991–997, 2002. leishmaniasis,” BMC Genomics, vol. 7, article 52, 2006. [115] D. V. Lovato, I. T. Nicolau de Campos, R. Amino, and A. [102] J. M. C. Ribeiro, R. Charlab, and J. G. Valenzuela, “The sali- S. Tanaka, “The full-length cDNA of anticoagulant protein vary adenosine deaminase activity of the mosquitoes Culex infestin revealed a novel releasable Kazal domain, a neu- quinquefasciatus and Aedes aegypti,” Journal of Experimental trophil elastase inhibitor lacking anticoagulant activity,” Biology, vol. 204, no. 11, pp. 2001–2010, 2001. Biochimie, vol. 88, no. 6, pp. 673–681, 2006. [103] R. Charlab, E. D. Rowton, and J. M. C. Ribeiro, “The [116] T. Friedrich, B. Kroger, S. Bialojan et al., “A Kazal-type salivary adenosine deaminase from the sand fly Lutzomyia inhibitor with thrombin specificity from Rhodnius prolixus,” longipalpis,” Experimental Parasitology, vol. 95, no. 1, pp. 45– The Journal of Biological Chemistry, vol. 268, no. 22, pp. 53, 2000. 16216–16222, 1993. [104] H. Kato, R. C. Jochim, P. G. Lawyer, and J. G. Valenzuela, [117] K. Mende, O. Petoukhova, V. Koulitchkova et al., “Dipetalo- “Identification and characterization of a salivary adenosine gastin, a potent thrombin inhibitor from the blood-sucking Psyche 15
insect Dipetalogaster maximus. cDNA cloning, expression [133] S. Schlehuber and A. Skerra, “Lipocalins in drug discovery: and characterization,” European Journal of Biochemistry, vol. from natural ligand-binding proteins to ’anticalins’,” Drug 266, no. 2, pp. 583–590, 1999. Discovery Today, vol. 10, no. 1, pp. 23–33, 2005. [118] H. Isawa, M. Yuda, K. Yoneda, and Y. Chinzei, “The insect [134] S. Ohno, Evolution by Gene Duplication, Springer, Berlin, salivary protein, prolixin-S, inhibits factor IXa generation Germany, 1970. and Xase complex formation in the blood coagulation [135] E. V.Koonin, “Orthologs, paralogs, and evolutionary genom- pathway,” The Journal of Biological Chemistry, vol. 275, no. ics,” Annual Review of Genetics, vol. 39, pp. 309–338, 2005. 9, pp. 6636–6641, 2000. [136] B. J. Mans, A. I. Louw, and A. W. H. Neitz, “Evolution of [119] H. J. Magert,¨ L. Standker,¨ P. Kreutzmann et al., “LEKTI, a hematophagy in ticks: common-origins for blood coagula- novel 15-domain type of human serine proteinase inhibitor,” tion and platelet aggregation inhibitors from soft ticks of the The Journal of Biological Chemistry, vol. 274, no. 31, pp. genus Ornithodoros,” Molecular Biology and Evolution, vol. 19, 21499–21502, 1999. no. 10, pp. 1695–1705, 2002. [120] R. Augustin, S. Siebert, and T. C. G. Bosch, “Identification [137] M. Hurles, “Gene duplication: the genomic trade in spare of a kazal-type serine protease inhibitor with potent anti- parts,” PLos Biology, vol. 2, no. 7, Article ID E206, 2004. staphylococcal activity as part of Hydra’s innate immune [138] J. M. C. Ribeiro, B. J. Mans, and B. Arca,` “An insight into the system,” Developmental and Comparative Immunology, vol. sialome of blood-feeding Nematocera,” Insect Biochemistry 33, no. 7, pp. 830–837, 2009. and Molecular Biology, vol. 40, no. 11, pp. 767–784, 2010. [121] P. Taka´c,ˇ M. A. Nunn, J. Mesz´ aros´ et al., “Vasotab, a vasoac- [139] B. G. Fry, K. Roelants, D. E. Champagne et al., “The tox- tive peptide from horse fly Hybomitra bimaculata (Diptera, icogenomic multiverse: convergent recruitment of proteins Tabanidae) salivary glands,” Journal of Experimental Biology, into animal venoms,” Annual Review of Genomics and Human vol. 209, no. 2, pp. 343–352, 2006. Genetics, vol. 10, pp. 483–511, 2009. [122] M. R. Kanost, “Serine proteinase inhibitors in arthropod [140] J. M. C Ribeiro and B. Arca, “From sialomes to the sialoverse: immunity,” Developmental and Comparative Immunology, an insight into the salivary potion of blood feeding insects,” vol. 23, no. 4-5, pp. 291–301, 1999. Advances in Insect Physiology, vol. 37, pp. 59–118, 2009. [123] J. C. Rau, L. M. Beaulieu, J. A. Huntington, and F. C. Church, [141] Y. Zhang, J. M. C. Ribeiro, J. A. Guimaraes,˜ and P. N. “Serpins in thrombosis, hemostasis and fibrinolysis,” Journal Walsh, “Nitrophorin-2: a novel mixed-type reversible specific of Thrombosis and Haemostasis, vol. 5, no. 1, pp. 102–115, inhibitor of the intrinsic factor-X activating complex,” Bio- 2007. chemistry, vol. 37, no. 30, pp. 10681–10690, 1998. [124] K. R. Stark and A. A. James, “Isolation and characterization [142] B. J. Mans and J. M. C. Ribeiro, “Function, mechanism and of the gene encoding a novel factor Xa-directed anticoagulant evolution of the moubatin-clade of soft tick lipocalins,” Insect from the yellow fever mosquito, Aedes aegypti,” The Journal of Biochemistry and Molecular Biology, vol. 38, no. 9, pp. 841– Biological Chemistry, vol. 273, no. 33, pp. 20802–20809, 1998. 852, 2008. [125] E. Calvo, D. M. Mizurini, A Sa-Nunes´ et al., “Alboserpin, a [143] B. J. Mans and J. M. C. Ribeiro, “A novel clade of cysteinyl factor Xa inhibitor from the mosquito vector of yellow fever, leukotriene scavengers in soft ticks,” Insect Biochemistry and binds heparin and membrane phospholipids and exhibits Molecular Biology, vol. 38, no. 9, pp. 862–870, 2008. antithrombotic activity,” The Journal of Biological Chemistry, [144] B. J. Mans, J. M. C. Ribeiro, and J. F. Andersen, “Structure, vol. 286, no. 32, pp. 27998–28010, 2011. function, and evolution of biogenic amine-binding proteins [126] C. Kellenberger and A. Roussel, “Structure-activity relation- in soft ticks,” The Journal of Biological Chemistry, vol. 283, no. ship within the serine protease inhibitors of the Pacifastin 27, pp. 18721–18733, 2008. family,” Protein and Peptide Letters, vol. 12, no. 5, pp. 409– [145] S. Sangamnatdej, G. C. Paesen, M. Slovak, and P. A. Nuttall, 414, 2005. “A high affinity serotonin- and histamine-binding lipocalin [127] M. Abrahamson, M. Alvarez-Fernandez, and C. M. from tick saliva,” Insect Molecular Biology, vol. 11, no. 1, pp. Nathanson, “Cystatins,” Biochemical Society Symposium,no. 79–86, 2002. 70, pp. 179–199, 2003. [146] G. C. Paesen, P. L. Adams, P. A. Nuttall, and D. L. Stuart, [128] M. Solomon, B. Belenghi, M. Delledonne, E. Menachem, “Tick histamine-binding proteins: lipocalins with a second and A. Levine, “The involvement of cysteine proteases and binding cavity,” Biochimica et Biophysica Acta, vol. 1482, no. protease inhibitor genes in the regulation of programmed cell 1-2, pp. 92–101, 2000. death in plants,” Plant Cell, vol. 11, no. 3, pp. 431–443, 1999. [147] G. C. Paesen, P. L. Adams, K. Harlos, P. A. Nuttall, and D. I. [129] M. Estelle, “Proteases and cellular regulation in plants,” Stuart, “Tick histamine-binding proteins: isolation, cloning, Current Opinion in Plant Biology, vol. 4, no. 3, pp. 254–260, and three-dimensional structure,” Molecular Cell, vol. 3, no. 2001. 5, pp. 661–671, 1999. [130] M. Kotsyfakis, A. Sa-Nunes,´ I. M. B. Francischetti, T. N. [148] M. A. Nunn, A. Sharma, G. C. Paesen et al., “Complement Mather, J. F. Andersen, and J. M. C. Ribeiro, “Antiinflam- inhibitor of C5 activation from the soft tick Ornithodoros matory and immunosuppressive activity of sialostatin L, a moubata,” Journal of Immunology, vol. 174, no. 4, pp. 2084– salivary cystatin from the tick Ixodes scapularis,” The Journal 2091, 2005. of Biological Chemistry, vol. 281, no. 36, pp. 26298–26307, [149] V. B. Wigglesworth et al., “The fate of haemoglobin in 2006. Rhodnius prolixus (Hemiptera) and other blood-sucking [131] D. R. Flower, A. C. T. North, and C. E. Sansom, “The lipocalin arthropods,” Proceedings of the Royal Society B, vol. 131, pp. protein family: structural and sequence overview,” Biochim- 313–339, 1942. ica et Biophysica Acta, vol. 1482, no. 1-2, pp. 9–24, 2000. [150] J. M. C. Ribeiro, M. Schneider, T. Isaias, J. Jurberg, C. Galvao,˜ [132] J. F. Andersen, N. P. Gudderra, I. M. B. Francischetti, and andJ.A.Guimaraes,˜ “Role of salivary antihemostatic com- J. M. C. Ribeiro, “The role of salivary lipocalins in blood ponents in blood feeding by triatomine bugs (Heteroptera),” feeding by Rhodnius prolixus,” Archives of Insect Biochemistry Journal of Medical Entomology, vol. 35, no. 4, pp. 599–610, and Physiology, vol. 58, no. 2, pp. 97–105, 2005. 1998. 16 Psyche
[151] K. Galindo and D. P. Smith, “A large family of divergent [166] J. Alves-Silva, J. M. C. Ribeiro, J. van den Abbeele et al., Drosophila odorant-binding proteins expressed in gustatory “An insight into the sialome of Glossina morsitans morsitans,” and olfactory sensilla,” Genetics, vol. 159, no. 3, pp. 1059– BMC Genomics, vol. 11, no. 1, article 213, 2010. 1072, 2001. [167] D. R. Maddison, K. S. Schulz, and W. P. Maddison, “The tree [152] D. S. Hekmat-Scafe, R. L. Dorit, and J. R. Carlson, “Molecular of life web project,” Zootaxa, no. 1668, pp. 19–40, 2007. evolution of odorant-binding protein genes OS-E and OS-F [168] C. Galvao,J.S.Patterson,D.DaSilvaRochaetal.,“Anew˜ in Drosophila,” Genetics, vol. 155, no. 1, pp. 117–127, 2000. species of Triatominae from Tamil Nadu, India,” Medical and [153] E. Calvo, B. J. Mans, J. M. C. Ribeiro, and J. F. Andersen, Veterinary Entomology, vol. 16, no. 1, pp. 75–82, 2002. “Multifunctionality and mechanism of ligand binding in a mosquito antiinflammatory protein,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 10, pp. 3728–3733, 2009. [154] B. J. Mans, E. Calvo, J. M. C. Ribeiro, and J. F. Andersen, “The crystal structure of D7r4, a salivary biogenic amine-binding protein from the malaria mosquito Anopheles gambiae,” The Journal of Biological Chemistry, vol. 282, no. 50, pp. 36626– 36633, 2007. [155] E. Calvo, B. J. Mans, J. F. Andersen, and J. M. C. Ribeiro, “Function and evolution of a mosquito salivary protein family,” The Journal of Biological Chemistry, vol. 281, no. 4, pp. 1935–1942, 2006. [156] H. Isawa, M. Yuda, Y. Orito, and Y. Chinzei, “A mosquito salivary protein inhibits activation of the plasma contact system by binding to factor XII and high molecular weight kininogen,” The Journal of Biological Chemistry, vol. 277, no. 31, pp. 27651–27658, 2002. [157] G. M. Gibbs, K. Roelants, and M. K. O’Bryan, “The CAP superfamily: cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins—roles in reproduction, cancer, and immune defense,” Endocrine Reviews, vol. 29, no. 7, pp. 865–897, 2008. [158] Y. Yamazaki and T. Morita, “Structure and function of snake venom cysteine-rich secretory proteins,” Toxicon, vol. 44, no. 3, pp. 227–231, 2004. [159] Y. Yamazaki, H. Koike, Y. Sugiyama et al., “Cloning and characterization of novel snake venom proteins that block smooth muscle contraction,” European Journal of Biochem- istry, vol. 269, no. 11, pp. 2708–2715, 2002. [160] M. Nobile, F. Noceti, G. Prestipino, and L. D. Possani, “Helothermine, a lizard venom toxin, inhibits calcium current in cerebellar granules,” Experimental Brain Research, vol. 110, no. 1, pp. 15–20, 1996. [161] M. Ameri, X. Wang, M. J. Wilkerson, M. R. Kanost, and A. B. Broce, “An immunoglobulin binding protein (antigen 5) of the stable fly (diptera: Muscidae) salivary gland stimulates bovine immune responses,” Journal of Medical Entomology, vol. 45, no. 1, pp. 94–101, 2008. [162] D. Ma, X. Xu, S. An et al., “A novel family of RGD- containing disintegrins (Tablysin-15) from the salivary gland of the horsefly tabanus yao targets αIIbβ3orαVβ3and inhibits platelet aggregation and angiogenesis,” Thrombosis and Haemostasis, vol. 105, no. 6, pp. 1032–1045, 2011. [163] D. Ma, Y. Wang, H. Yang et al., “Anti-thrombosis repertoire of blood-feeding horsefly salivary glands,” Molecular and Cellular Proteomics, vol. 8, no. 9, pp. 2071–2079, 2009. [164] X. Xu, H. Yang, D. Ma et al., “Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands,” Molecular and Cellular Proteomics, vol. 7, no. 3, pp. 582–590, 2008. [165] D. C. de Graaf, M. Aerts, M. Brunain et al., “Insights into the venom composition of the ectoparasitoid wasp Nasonia vitripennis from bioinformatic and proteomic studies,” Insect Molecular Biology, vol. 19, no. 1, pp. 11–26, 2010. Copyright of Psyche: A Journal of Entomology is the property of Hindawi Publishing Corporation and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.