The Nematocera infraorder Culicomorpha is believed to have descended from bloodfeeding ancestors over 200 million years ago, generating bloodfeeding and non-bloodfeeding flies in two superfamilies, the Culicoidea-containing the mosquitoes, the frog-feeding midges, the Chaoboridae, and the Dixidae-and the Chironomoidea-containing the black flies, the ceratopogonids, the Chironomidae, and the Thaumaleidae. Blood feeding requires many adaptations, including development of a sophisticated salivary potion that disarms host hemostasis, the physiologic mechanism comprising platelet aggregation, vasoconstriction, and blood clotting. The composition of the sialome (from the Greek sialo = saliva) from bloodfeeding animals can be inferred from analysis of their salivary gland transcriptome. While members of the mosquitoes, black flies, and biting midges have provided sialotranscriptome descriptions, no species of the frog-biting midges has been thus analyzed. We describe in this work the sialotranscriptome of Corethrella appendiculata, revealing a complex potion of enzymes, classical nematoceran protein families involved in blood feeding, and novel protein families unique to this species of frog-feeding fly. Bacterial (Wolbachia) and novel viral sequences were also discovered.

Triatoma matogrossensis is a Hemiptera that belongs to the oliveirai complex, a vector of Chagas' disease that feeds on vertebrate blood in all life stages. Hematophagous insects' salivary glands (SGs) produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. Exposure to T. matogrossensis was also found to be a risk factor associated with the endemic form of the autoimmune skin disease pemphigus foliaceus, which is described in the same regions where Chagas' disease is observed in Brazil. To obtain a further insight into the salivary biochemical and pharmacologic diversity of this kissing bug and to identify possible allergens that might be associated with this autoimmune disease, a cDNA library from its SGs was randomly sequenced. We present the analysis of a set of 2,230 (SG) cDNA sequences, 1,182 of which coded for proteins of a putative secretory nature.

The kissing bug Triatoma rubida (Uhler, 1894) is found in southwestern United States and parts of Mexico where it is found infected with Trypanosoma cruzi, invades human dwellings and causes allergies from their bites. Although the protein salivary composition of several triatomine species is known, not a single salivary protein sequence is known from T. rubida. Furthermore, the salivary diversity of related hematophagous arthropods is very large probably because of the immune pressure from their hosts. Here we report the sialotranscriptome analysis of T. rubida based on the assembly of 1,820 high-quality expressed sequence tags, 51% of which code for putative secreted peptides, including lipocalins, members of the antigen five family, apyrase, hemolysin, and trialysin families. Interestingly, T. rubida lipocalins are at best 40% identical in primary sequence to those of T. protracta, a kissing bug that overlaps its range with T. rubida, indicating the diversity of the salivary lipocalins among species of the same hematophagous genus. We additionally found several expressed sequence tags coding for proteins of clear Trypanosoma spp. origin. This work contributes to the future development of markers of human and pet exposure to T. rubida and to the possible development of desensitization therapies. Supp. Data 1 and 2 (online only) of the transcriptome and deducted protein sequences can be obtained from http://exon.niaid.nih.gov/transcriptome/Trubida/Triru-S1-web.xlsx and http://exon.niaid.nih.gov/transcriptome/Trubida/Triru-S2-web.xlsx.

Background Saliva of hematophagous arthropods contains a diverse mixture of compounds that counteracts host hemostasis. Immunomodulatory and antiinflammatory components are also found in these organisms' saliva. Blood feeding evolved at least ten times within arthropods, providing a scenario of convergent evolution for the solution of the salivary potion. Perhaps because of immune pressure from hosts, the salivary proteins of related organisms have considerable divergence, and new protein families are often found within different genera of the same family or even among subgenera. Fleas radiated with their vertebrate hosts, including within the mammal expansion initiated 65 million years ago. Currently, only one flea species–the rat flea Xenopsylla cheopis–has been investigated by means of salivary transcriptome analysis to reveal salivary constituents, or sialome. We present the analysis of the sialome of cat flea Ctenocephaides felis. Methodology and Critical Findings A salivary gland cDNA library from adult fleas was randomly sequenced, assembled, and annotated. Sialomes of cat and rat fleas have in common the enzyme families of phosphatases (inactive), CD-39-type apyrase, adenosine deaminases, and esterases. Antigen-5 members are also common to both sialomes, as are defensins. FS-I/Cys7 and the 8-Cys families of peptides are also shared by both fleas and are unique to these organisms. The Gly-His-rich peptide similar to holotricin was found only in the cat flea, as were the abundantly expressed Cys-less peptide and a novel short peptide family. Conclusions/Significance Fleas, in contrast to bloodsucking Nematocera (mosquitoes, sand flies, and black flies), appear to concentrate a good portion of their sialome in small polypeptides, none of which have a known function but could act as inhibitors of hemostasis or inflammation. They are also unique in expansion of a phosphatase family that appears to be deficient of enzyme activity and has an unknown function.

Among several pharmacological compounds, Phlebotomine saliva contains substances with anti-inflammatory properties. In this article, we demonstrated the therapeutic activity of salivary gland extract (SGE) of Phlebotomus papatasi in an experimental model of arthritis (collagen-induced arthritis [CIA]) and identified the constituents responsible for such activity. Daily administration of SGE, initiated at disease onset, attenuated the severity of CIA, reducing the joint lesion and proinflammatory cytokine release. In vitro incubation of dendritic cells (DCs) with SGE limited specific CD4+ Th17 cell response. We identified adenosine (ADO) and 5′AMP as the major salivary molecules responsible for anti-inflammatory activities. Pharmacologic inhibition of ADO A2A receptor or enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect. Importantly, CD73 (ecto-5′-nucleotidase enzyme) is expressed on DC surface during stage of activation, suggesting that ADO is also generated by 5′AMP metabolism. Moreover, both nucleosides mimicked SGE-induced anti-inflammatory activity upon DC function in vitro and attenuated establishment of CIA in vivo. We reveal that ADO and 5′AMP are present in pharmacological amounts in P. papatasi saliva and act preferentially on DC function, consequently reducing Th17 subset activation and suppressing the autoimmune response. Thus, it is plausible that these constituents might be promising therapeutic molecules to target immune inflammatory diseases.

Ticks are mites specialized in acquiring blood from vertebrates as their sole source of food and are important disease vectors to humans and animals. Among the specializations required for this peculiar diet, ticks evolved a sophisticated salivary potion that can disarm their host's hemostasis, inflammation, and immune reactions. Previous transcriptome analysis of tick salivary proteins has revealed many new protein families indicative of fast evolution, possibly due to host immune pressure. The hard ticks (family Ixodidae) are further divided into two basal groups, of which the Metastriata have 11 genera. While salivary transcriptomes and proteomes have been described for some of these genera, no tick of the genus Hyalomma has been studied so far. The analysis of 2084 expressed sequence tags (EST) from a salivary gland cDNA library allowed an exploration of the proteome of this tick species by matching peptide ions derived from MS/MS experiments to this data set. We additionally compared these MS/MS derived peptide sequences against the proteins from the bovine host, finding many host proteins in the salivary glands of this tick. This annotated data set can assist the discovery of new targets for anti-tick vaccines as well as help to identify pharmacologically active proteins.

LJM11, an abundant salivary protein from the sand fly Lutzomyia longipalpis, belongs to the insect "yellow" family of proteins. In this study, we immunized mice with 17 plasmids encoding L. longiplapis salivary proteins and demonstrated that LJM11 confers protective immunity against Leishmania major infection. This protection correlates with a strong induction of a delayed type hypersensitivity (DTH) response following exposure to L. longipalpis saliva. Additionally, splenocytes of exposed mice produce IFN-γ upon stimulation with LJM11, demonstrating the systemic induction of Th1 immunity by this protein. In contrast to LJM11, LJM111, another yellow protein from L. longipalpis saliva, does not produce a DTH response in these mice, suggesting that structural or functional features specific to LJM11 are important for the induction of a robust DTH response. To examine these features, we used calorimetric analysis to probe a possible ligand binding function for the salivary yellow proteins. LJM11, LJM111, and LJM17 all acted as high affinity binders of prohemostatic and proinflammatory biogenic amines, particularly serotonin, catecholamines, and histamine. We also determined the crystal structure of LJM11, revealing a six-bladed β-propeller fold with a single ligand binding pocket located in the central part of the propeller structure on one face of the molecule. A hypothetical model of LJM11 suggests a positive electrostatic potential on the face containing entry to the ligand binding pocket, whereas LJM111 is negative to neutral over its entire surface. This may be the reason for differences in antigenicity between the two proteins.

Dipetalogaster maxima is a blood-sucking Hemiptera that inhabits sylvatic areas in Mexico. It usually takes its blood meal from lizards, but following human population growth, it invaded suburban areas, feeding also on humans and domestic animals. Hematophagous insect salivary glands produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain further insight into the salivary biochemical and pharmacologic complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Salivary proteins were also submitted to one- and two-dimensional gel electrophoresis (1DE and 2DE) followed by mass spectrometry analysis. We present the analysis of a set of 2728 cDNA sequences, 1375 of which coded for proteins of a putative secretory nature. The saliva 2DE proteome displayed approximately 150 spots. The mass spectrometry analysis revealed mainly lipocalins, pallidipins, antigen 5-like proteins, and apyrases. The redundancy of sequence identification of saliva-secreted proteins suggests that proteins are present in multiple isoforms or derive from gene duplications.

Saliva of blood-sucking arthropods contains a complex mixture of peptides that affect their host's hemostasis, inflammation, and immunity. These activities can also modify the site of pathogen delivery and increase disease transmission. Saliva also induces hosts to mount an antisaliva immune response that can lead to skin allergies or even anaphylaxis. Accordingly, knowledge of the salivary repertoire, or sialome, of a mosquito is useful to provide a knowledge platform to mine for novel pharmacological activities, to develop novel vaccine targets for vector-borne diseases, and to develop epidemiological markers of vector exposure and candidate desensitization vaccines. The mosquito Ochlerotatus triseriatus is a vector of La Crosse virus and produces allergy in humans. In this work, a total of 1,575 clones randomly selected from an adult female O. triseriatus salivary gland cDNA library was sequenced and used to assemble a database that yielded 731 clusters of related sequences, 560 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 159 protein sequences, 66 of which code for putative secreted proteins. Supplemental spreadsheets containing these data are available at http://exon.niaid.nih.gov/transcriptome/Ochlerotatus_triseriatus/S1/Ot-S1.xls and http://exon.niaid. nih.gov/transcriptome/Ochlerotatus_triseriatus/S2/Ot-S2.xls.

The evolution of insects to a blood diet leads to the development of a saliva that antagonizes their hosts' hemostasis and inflammation. Hemostasis and inflammation are redundant processes, and thus a complex salivary potion composed of dozens or near 100 different polypeptides is commonly found by transcriptome or proteome analysis of these organisms. Several insect orders or families evolved independently to hematophagy, creating unique salivary potions in the form of novel pharmacological use of endogenous substances and in the form of unique proteins not matching other known proteins, these probably arriving by fast evolution of salivary proteins as they evade their hosts' immune response. In this work we present a preliminary description of the sialome (from the Greek Sialo = saliva) of the common bed bug Cimex lectularius, the first such work from a member of the Cimicidae family. This manuscript is a guide for the supplemental database files http://exon.niaid.nih.gov/transcriptome/C_lectularius/S1/Cimex-S1.zip and http://exon.niaid.nih.gov/transcriptome/C_lectularius/S2/Cimex-S2.xls

Background: Saliva of adult female mosquitoes help sugar and blood feeding by providing enzymes and polypeptides that help sugar digestion, control microbial growth and counteract their vertebrate host hemostasis and inflammation. Mosquito saliva also potentiates the transmission of vector borne pathogens, including arboviruses. Culex tarsalis is a bird feeding mosquito vector of West Nile Virus closely related to C. quinquefasciatus, a mosquito relatively recently adapted to feed on humans, and the only mosquito of the genus Culex to have its sialotranscriptome so far described.Results: A total of 1,753 clones randomly selected from an adult female C. tarsalis salivary glands (SG) cDNA library were sequenced and used to assemble a database that yielded 809 clusters of related sequences, 675 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 283 protein sequences, 80 of which code for putative secreted proteins.Conclusion: Comparison of the C. tarsalis sialotranscriptome with that of C. quinquefasciatus reveals accelerated evolution of salivary proteins as compared to housekeeping proteins. The average amino acid identity among salivary proteins is 70.1%, while that for housekeeping proteins is 91.2% (P < 0.05), and the codon volatility of secreted proteins is significantly higher than those of housekeeping proteins. Several protein families previously found exclusive of mosquitoes, including only in the Aedes genus have been identified in C. tarsalis. Interestingly, a protein family so far unique to C. quinquefasciatus, with 30 genes, is also found in C. tarsalis, indicating it was not a specific C. quinquefasciatus acquisition in its evolution to optimize mammal blood feeding.

Pemphigus foliaceus is a life threatening skin disease that is associated with autoimmunity to desmoglein, a skin protein involved in the adhesion of keratinocytes. This disease is endemic in certain areas of South America, suggesting the mediation of environmental factors triggering autoimmunity. Among the possible environmental factors, exposure to bites of black flies, in particular Simulium nigrimanum has been suggested. In this work, we describe the sialotranscriptome of adult female S. nigrimanum flies. It reveals the complexity of the salivary potion of this insect, comprised by over 70 distinct genes within over 30 protein families, including several novel families, even when compared with the previously described sialotranscriptome of the autogenous black fly, S. vittatum. The uncovering of this sialotranscriptome provides a platform for testing pemphigus patient sera against recombinant salivary proteins from S. nigrimanum and for the discovery of novel pharmacologically active compounds.

Adaptation to vertebrate blood feeding includes development of a salivary "magic potion" that can disarm host hemostasis and inflammatory reactions. Within the lower Diptera, a vertebrate blood-sucking mode evolved in the Psychodidae (sand flies), Culicidae (mosquitoes), Ceratopogonidae (biting midges), Simuliidae (black flies), and the frog-feeding Corethrellidae. Sialotranscriptome analyses from several species of mosquitoes and sand flies and from one biting midge indicate divergence in the evolution of the blood-sucking salivary potion, manifested in the finding of many unique proteins within each insect family, and even genus. Gene duplication and divergence events are highly prevalent, possibly driven by vertebrate host immune pressure. Within this framework, we describe the sialome (from Greek sialo, saliva) of the black fly Simulium vittatum and discuss the findings within the context of the protein families found in other blood-sucking Diptera. Sequences and results of Blast searches against several protein family databases are given in Supplemental Tables S1 and S2, which can be obtained from http://exon.niaid.nih.gov/transcriptome/S_vittatum/T1/SV-tb1.zip and http://exon.niaid.nih.gov/transcriptome/S_vittatum/T2/SV-tb2.zip .

BACKGROUND:Mosquito saliva, consisting of a mixture of dozens of proteins affecting vertebrate hemostasis and having sugar digestive and antimicrobial properties, helps both blood and sugar meal feeding. Culicine and anopheline mosquitoes diverged ~150 MYA, and within the anophelines, the New World species diverged from those of the Old World ~95 MYA. While the sialotranscriptome (from the Greek sialo, saliva) of several species of the Cellia subgenus of Anopheles has been described thoroughly, no detailed analysis of any New World anopheline has been done to date. Here we present and analyze data from a comprehensive salivary gland (SG) transcriptome of the neotropical malaria vector Anopheles darlingi (subgenus Nyssorhynchus).RESULTS:A total of 2,371 clones randomly selected from an adult female An. darlingi SG cDNA library were sequenced and used to assemble a database that yielded 966 clusters of related sequences, 739 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 183 protein sequences, 114 of which code for putative secreted proteins.CONCLUSION:Comparative analysis of sialotranscriptomes of An. darlingi and An. gambiae reveals significant divergence of salivary proteins. On average, salivary proteins are only 53% identical, while housekeeping proteins are 86% identical between the two species. Furthermore, An. darlingi proteins were found that match culicine but not anopheline proteins, indicating loss or rapid evolution of these proteins in the old world Cellia subgenus. On the other hand, several well represented salivary protein families in old world anophelines are not expressed in An. darlingi.

The salivary glands of blood-sucking arthropods contain a redundant 'magic potion' that counteracts their vertebrate host's hemostasis, inflammation, and immunity. We here describe the salivary transcriptome and proteomics (sialome) of the soft tick Ornithodoros coriaceus. The resulting analysis helps to consolidate the classification of common proteins found in both soft and hard ticks, such as the lipocalins, Kunitz, cystatin, basic tail, hebraein, defensin, TIL domain, metalloprotease, 5'-nucleotidase/apyrase, and phospholipase families, and also to identify protein families uniquely found in the Argasidae, such as the adrenomedullin/CGRP peptides, 7DB, 7 kDa, and the RGD-containing single-Kunitz proteins. Additionally, we found a protein belonging to the cytotoxin protein family that has so far only been identified in hard ticks. Three other unique families common only to the Ornithodoros genus were discovered. Edman degradation, 2D and 1D-PAGE of salivary gland homogenates followed by tryptic digestion and HPLC MS/MS of results confirms, the presence of several proteins. These results indicate that each genus of hematophagous arthropods studied to date evolved unique protein families that assist blood feeding, thus characterizing potentially new pharmacologically active components or antimicrobial agents. Published by Elsevier B.V.