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1.  Simplagrin, a Platelet Aggregation Inhibitor from Simulium nigrimanum Salivary Glands Specifically Binds to the Von Willebrand Factor Receptor in Collagen and Inhibits Carotid Thrombus Formation In Vivo 
Among the several challenges faced by bloodsucking arthropods, the vertebrate hemostatic response against blood loss represents an important barrier to efficient blood feeding. Here we report the first inhibitor of collagen-induced platelet aggregation derived from the salivary glands of a black fly (Simulium nigrimanum), named Simplagrin.
Methods and Findings
Simplagrin was expressed in mammalian cells and purified by affinity-and size-exclusion chromatography. Light-scattering studies showed that Simplagrin has an elongated monomeric form with a hydrodynamic radius of 5.6 nm. Simplagrin binds to collagen (type I-VI) with high affinity (2–15 nM), and this interaction does not involve any significant conformational change as determined by circular dichroism spectroscopy. Simplagrin-collagen interaction is both entropically and enthalpically driven with a large negative ΔG, indicating that this interaction is favorable and occurs spontaneously. Simplagrin specifically inhibits von Willebrand factor interaction with collagen type III and completely blocks platelet adhesion to collagen under flow conditions at high shear rates; however, Simplagrin failed to block glycoprotein VI and Iα2β1 interaction to collagen. Simplagrin binds to RGQOGVMGF peptide with an affinity (KD 11 nM) similar to that of Simplagrin for collagen. Furthermore, Simplagrin prevents laser-induced carotid thrombus formation in vivo without significant bleeding in mice and could be useful as an antithrombotic agent in thrombosis related disease.
Our results support the orthology of the Aegyptin clade in bloodsucking Nematocera and the hypothesis of a faster evolutionary rate of salivary function of proteins from blood feeding arthropods.
Author Summary
Blood feeding arthropods—like mosquitoes and black flies—have evolved salivary secretions rich in molecules that affect hemostasis, including vasodilators and inhibitors of blood clotting and platelet aggregation. Among the platelet inhibitors, antagonists of collagen-induced platelet aggregation and adhesion have been found in salivary glands of blood feeders. Here we report the first collagen-binding protein from salivary glands of a black fly. This molecule prevents thrombosis in mice without causing significant bleeding, making it an attractive candidate as an antithrombotic agent. Because blackflies and mosquitoes shared a common blood feeding ancestor approximately 250 million years ago, it appears that collagen-binding activity in salivary glands was an evolutionary innovation present in an ancient dipteran ancestor. Our work highlights the central role of inhibition of platelet aggregation as a vital salivary function in blood feeding arthropods.
PMCID: PMC4055580  PMID: 24921659
2.  Lundep, a Sand Fly Salivary Endonuclease Increases Leishmania Parasite Survival in Neutrophils and Inhibits XIIa Contact Activation in Human Plasma 
PLoS Pathogens  2014;10(2):e1003923.
Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET) were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep) from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep) shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate)- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized) Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.
Author Summary
Salivary components from disease vectors help the arthropod to acquire blood. Here we show that an arthropod vector salivary enzyme affects the innate immune system of the host—mainly the destruction of neutrophil traps—allowing the Leishmania parasite to evade the host immune response and to cause an infection. This work highlights the relevance of vector salivary components in parasite transmission and further suggests the inclusion of these proteins as components for an anti-Leishmania vaccine. Importantly, because salivary proteins are always present at the site of natural transmission, this work further encourages the testing of vaccine candidates using the natural route of transmission—the bites of an arthropod vector—instead of current practices based solely on needle injection of parasites.
PMCID: PMC3916414  PMID: 24516388
3.  A deep insight into the sialotranscriptome of the mosquito, Psorophora albipes 
BMC Genomics  2013;14:875.
Psorophora mosquitoes are exclusively found in the Americas and have been associated with transmission of encephalitis and West Nile fever viruses, among other arboviruses. Mosquito salivary glands represent the final route of differentiation and transmission of many parasites. They also secrete molecules with powerful pharmacologic actions that modulate host hemostasis, inflammation, and immune response. Here, we employed next generation sequencing and proteome approaches to investigate for the first time the salivary composition of a mosquito member of the Psorophora genus. We additionally discuss the evolutionary position of this mosquito genus into the Culicidae family by comparing the identity of its secreted salivary compounds to other mosquito salivary proteins identified so far.
Illumina sequencing resulted in 13,535,229 sequence reads, which were assembled into 3,247 contigs. All families were classified according to their in silico-predicted function/ activity. Annotation of these sequences allowed classification of their products into 83 salivary protein families, twenty (24.39%) of which were confirmed by our subsequent proteome analysis. Two protein families were deorphanized from Aedes and one from Ochlerotatus, while four protein families were described as novel to Psorophora genus because they had no match with any other known mosquito salivary sequence. Several protein families described as exclusive to Culicines were present in Psorophora mosquitoes, while we did not identify any member of the protein families already known as unique to Anophelines. Also, the Psorophora salivary proteins had better identity to homologs in Aedes (69.23%), followed by Ochlerotatus (8.15%), Culex (6.52%), and Anopheles (4.66%), respectively.
This is the first sialome (from the Greek sialo = saliva) catalog of salivary proteins from a Psorophora mosquito, which may be useful for better understanding the lifecycle of this mosquito and the role of its salivary secretion in arboviral transmission.
PMCID: PMC3878727  PMID: 24330624
4.  Effects of Aedes aegypti salivary components on dendritic cell and lymphocyte biology 
Parasites & Vectors  2013;6:329.
Saliva is a key element of interaction between hematophagous mosquitoes and their vertebrate hosts. In addition to allowing a successful blood meal by neutralizing or delaying hemostatic responses, the salivary cocktail is also able to modulate the effector mechanisms of host immune responses facilitating, in turn, the transmission of several types of microorganisms. Understanding how the mosquito uses its salivary components to circumvent host immunity might help to clarify the mechanisms of transmission of such pathogens and disease establishment.
Flow cytometry was used to evaluate if increasing concentrations of A. aegypti salivary gland extract (SGE) affects bone marrow-derived DC differentiation and maturation. Lymphocyte proliferation in the presence of SGE was estimated by a colorimetric assay. Western blot and Annexin V staining assays were used to assess apoptosis in these cells. Naïve and memory cells from mosquito-bite exposed mice or OVA-immunized mice and their respective controls were analyzed by flow cytometry.
Concentration-response curves were employed to evaluate A. aegypti SGE effects on DC and lymphocyte biology. DCs differentiation from bone marrow precursors, their maturation and function were not directly affected by A. aegypti SGE (concentrations ranging from 2.5 to 40 μg/mL). On the other hand, lymphocytes were very sensitive to the salivary components and died in the presence of A. aegypti SGE, even at concentrations as low as 0.1 μg/mL. In addition, A. aegypti SGE was shown to induce apoptosis in all lymphocyte populations evaluated (CD4+ and CD8+ T cells, and B cells) through a mechanism involving caspase-3 and caspase-8, but not Bim. By using different approaches to generate memory cells, we were able to verify that these cells are resistant to SGE effects.
Our results show that lymphocytes, and not DCs, are the primary target of A. aegypti salivary components. In the presence of A. aegypti SGE, naïve lymphocyte populations die by apoptosis in a caspase-3- and caspase-8-dependent pathway, while memory cells are selectively more resistant to its effects. The present work contributes to elucidate the activities of A. aegypti salivary molecules on the antigen presenting cell-lymphocyte axis and in the biology of these cells.
PMCID: PMC3843549  PMID: 24238038
Dendritic cells; T cells; Aedes aegypti; Saliva; Apoptosis
5.  Tick salivary secretion as a source of antihemostatics 
Journal of Proteomics  2012;75(13):3842-3854.
Ticks are mostly obligatory blood feeding ectoparasites that have an impact on human and animal health. In addition to direct damage due to feeding, some tick species serve as the vectors for the causative agents of several diseases, such as the spirochetes of the genus Borrelia causing Lyme disease, the virus of tick-borne encephalitis, various Rickettsial pathogens or even protozoan parasites like Babesia spp. Hard ticks are unique among bloodfeeders because of their prolonged feeding period that may last up to two weeks. During such a long period of blood uptake, the host develops a wide range of mechanisms to prevent blood loss. The arthropod ectoparasite, in turn, secretes saliva in the sites of bite that assists blood feeding. Indeed, tick saliva represents a rich source of proteins with potent pharmacologic action that target different mechanisms of coagulation, platelet aggregation and vasoconstriction. Tick adaptation to their vertebrate hosts led to the inclusion of a powerful protein armamentarium in their salivary secretion that has been investigated by high throughput methods. The resulting knowledge can be exploited for the isolation of novel antihemostatic agents. Here we review the tick salivary antihemostatics and their characterized functions at the molecular and cellular levels.
PMCID: PMC3383439  PMID: 22564820
tick; salivary gland; hemostasis; coagulation; platelet aggregation; serine proteases; thrombin; kunitz-domain protein; disintegrin; serpin
6.  Cysteine Proteases from Bloodfeeding Arthropod Ectoparasites 
Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion, and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells.
PMCID: PMC3413451  PMID: 21660665
activity-based probes; arthropod; aspartic aminopeptidase; Babesia; blood feeding; caspase; cathepsin-B; cathepsin-C; cathepsin-D; cathepsin-L; cysteine proteases; ecdysone; ectoparasite; embryogenesis; fat body; hematophagy; hemoglobin; legumain RNAi; leucine aminopeptidase; lysosome; malaria; metamorphosis; midgut; mosquito; oocyte; Plasmodium; tick; vitellin; yolk protein
7.  The Salivary Gland Transcriptome of the Eastern Tree Hole Mosquito, Ochlerotatus triseriatus 
Journal of Medical Entomology  2010;47(3):376-386.
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 and
PMCID: PMC3394432  PMID: 20496585
mosquito; salivary gland; sialome; transcriptome; La Crosse virus
8.  An insight into the sialome of Simulium guianense (DIPTERA:SIMulIIDAE), the main vector of River Blindness Disease in Brazil 
BMC Genomics  2011;12:612.
Little is known about the composition and function of the saliva in black flies such as Simulium guianense, the main vector of river blindness disease in Brazil. The complex salivary potion of hematophagous arthropods counteracts their host's hemostasis, inflammation, and immunity.
Transcriptome analysis revealed ubiquitous salivary protein families--such as the Antigen-5, Yellow, Kunitz domain, and serine proteases--in the S. guianense sialotranscriptome. Insect-specific families were also found. About 63.4% of all secreted products revealed protein families found only in Simulium. Additionally, we found a novel peptide similar to kunitoxin with a structure distantly related to serine protease inhibitors. This study revealed a relative increase of transcripts of the SVEP protein family when compared with Simulium vittatum and S. nigrimanum sialotranscriptomes. We were able to extract coding sequences from 164 proteins associated with blood and sugar feeding, the majority of which were confirmed by proteome analysis.
Our results contribute to understanding the role of Simulium saliva in transmission of Onchocerca volvulus and evolution of salivary proteins in black flies. It also consists of a platform for mining novel anti-hemostatic compounds, vaccine candidates against filariasis, and immuno-epidemiologic markers of vector exposure.
PMCID: PMC3285218  PMID: 22182526
9.  An insight into the sialome of the bed bug, Cimex lectularius 
Journal of proteome research  2010;9(8):3820-3831.
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 comprised of dozens or near one hundred 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 and
PMCID: PMC2917537  PMID: 20441151
Bedbug; saliva; salivary transcriptome; salivary proteome
10.  Aegyptin displays high-affinity for Willebrand Factor Binding Site (RGQOGVMGF) in Collagen and Inhibits Carotid Thrombus Formation in vivo 
The FEBS journal  2009;277(2):413-427.
Aegyptin is a 30-kDa mosquito salivary gland protein that binds to collagen and inhibits platelet aggregation. We have studied biophysical properties of aegyptin and its mechanism of action. Light scattering plot shows that aegyptin displays a monomeric elongated form which explains the apparent molecular mass of 110 kDa estimated by gel-filtration chromatography. Surface plasmon resonance identified the sequence RGQOGVMGF (O is hydroxyproline) that mediates collagen interaction with von Willebrand Factor (vWF) as high-affinity (KD ≈ 10 nM) binding site for aegyptin. Additionally, aegyptin interacts with linear RGQPGVMGF peptide and heat-denatured collagen, implying that the triple-helix and hydroxyproline are not a prerequisite for binding. In contrast, aegyptin does not interact with scrambled RGQPGVMGF peptide. Aegyptin also recognizes with low affinity (µM range) peptides (GPO)10 and GFOGER which respectively represent glycoprotein VI and integrin α2β1 binding sites in collagen, and prevents platelet adhesion and aggregation. Truncated forms of aegyptin were engineered, and the C-terminus fragment was shown to interact with collagen and to attenuate platelet aggregation. In addition, aegyptin prevents laser-induced carotid thrombus formation in the presence of Rose Bengal in vivo, without observable bleeding in rats. In conclusion, aegyptin interacts with distinct binding sites in collagen, and is useful tool to inhibit platelet-collagen interaction in vitro and in vivo.
PMCID: PMC2892622  PMID: 20015075
aegyptin; blood-sucking; hematophagy; mosquitoes; yellow fever; GPVI
11.  The Function and Three-Dimensional Structure of a Thromboxane A2/Cysteinyl Leukotriene-Binding Protein from the Saliva of a Mosquito Vector of the Malaria Parasite 
PLoS Biology  2010;8(11):e1000547.
A salivary protein from a malaria-transmitting mosquito uses a single domain to bind to thromboxane A2 and cysteinyl leukotrienes and prevent blood clotting and inflammation in the host on which it feeds.
The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A2 (TXA2) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C4 (LTC4)-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA2 analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD7. In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA2 analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA2 analog U46619 is stabilized by hydrogen bonding interactions of the ω-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC4 and occupies a very similar position to LTE4 in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.
Author Summary
When feeding, a female mosquito must inhibit the blood clotting and inflammatory responses of the host. To do this, the insect produces salivary proteins that neutralize key host molecules participating in clotting and inflammation. Here, we describe a salivary protein AnSt-D7L1 that scavenges both thomboxane A2 and cysteinyl leukotrienes, two substances involved in blood vessel constriction, platelet aggregation, and inflammatory responses to an insect bite. We produced this protein in bacteria and showed that it tightly binds both these molecules, inhibiting the processes in which they are involved. We then determined its structure using X-ray crystallography and showed that there is a single binding site in one domain of the protein, accommodating both thromboxane A2 and cysteinyl leukotrienes, and that this site is responsible for the scavenging effect of the protein. These studies reveal the structural features of proteins needed to bind to key molecules of potential pharmacological importance and add to our understanding of the process of mosquito blood feeding, which is essential for transmission of the malaria parasite.
PMCID: PMC2994686  PMID: 21152418
12.  Aegyptin, a Novel Mosquito Salivary Gland Protein Specifically Binds to Collagen and Prevents its Interaction with Glycoprotein VI, Integrin α2β1 and von Willebrand Factor 
The Journal of biological chemistry  2007;282(37):26928-26938.
Blood-sucking arthropods have evolved a number of inhibitors of platelet aggregation and blood coagulation. In this report we have molecularly and functionally characterized aegyptin, a member of the family of 30-kDa salivary allergens from Aedes aegypti, whose function remained elusive thus far. Aegyptin displays a unique sequence characterized by glycine, glutamic acid, and aspartic acid repeats and was shown to specifically block collagen-induced human platelet aggregation and granule secretion. Plasmon resonance experiments demonstrate that aegyptin binds to collagen types I-V (Kd ≈ 1 nM) but does not interact with vitronectin, fibronectin, laminin, fibrinogen, and von Willebrand factor (vWf). In addition, aegyptin attenuates platelet adhesion to soluble or fibrillar collagen. Furthermore, aegyptin inhibits vWf interaction with collagen type III under static conditions and completely blocks platelet adhesion to collagen under flow conditions at high shear rates. Notably, aegyptin completely prevents collagen but not convulxin binding to recombinant glycoprotein VI. These findings indicate that aegyptin recognizes specific binding sites for glycoprotein VI, integrin α2β1, and vWf, thereby preventing collagen interaction with its three major ligands. Aegyptin is a novel tool to study collagen-platelet interaction and a prototype for development of molecules with antithrombotic properties.
PMCID: PMC2913440  PMID: 17650501
13.  An insight into the sialotranscriptome of the West Nile mosquito vector, Culex tarsalis 
BMC Genomics  2010;11:51.
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.
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.
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.
PMCID: PMC2823692  PMID: 20089177
14.  Immunity against a ‘silent’ salivary antigen of the Lyme vector Ixodes scapularis impairs its ability to feed 
Ixodes scapularis ticks transmit the Lyme disease agent in the US. Although strong anti-tick immunity mediates tick rejection by certain vertebrates, only a few antigens have been molecularly characterized. We show that guinea pig vaccination against a secreted tick salivary immunomodulator—sialostatin L2—can lead to decreased feeding ability of I. scapularis nymphs. Increased rejection rate, prolonged feeding time and apparent signs of inflammation were observed for nymphs attached to vaccinated animals, indicating a protective host immune response. Interestingly, sialostatin L2 humoral recognition does not take place upon repeated tick exposure in control animals, but only in the vaccinated animals that neutralize sialostatin L2 action. Therefore, we demonstrate an essential sialostatin L2 role upon nymphal infestation that can be blocked by vertebrate immunity and we propose the discovery of similarly ‘silent’ antigens towards the development of a multicomponent vaccine that will protect against tick bites and the pathogens they transmit.
PMCID: PMC2562228  PMID: 18832673
‘silent’ antigens; immunity; protection; vaccine; vector
15.  The salivary gland transcriptome of the neotropical malaria vector Anopheles darlingi reveals accelerated evolution of genes relevant to hematophagy 
BMC Genomics  2009;10:57.
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).
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.
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.
PMCID: PMC2644710  PMID: 19178717
16.  An insight into the sialome of Anopheles funestus reveals an emerging pattern in anopheline salivary protein families 
Anopheles funestus, together with Anopheles gambiae, is responsible for most malaria transmission in sub-Saharan Africa, but little is known about molecular aspects of its biology. To investigate the salivary repertoire of this mosquito, we randomly sequenced 916 clones from a salivary-gland cDNA library from adult female F1 offspring of field-caught An. funestus. Thirty-three protein sequences, mostly full-length transcripts, are predicted to be secreted salivary proteins. We additionally describe 25 full-length housekeeping-associated transcripts. In accumulating mosquito sialotranscriptome information—which includes An. gambiae, Anopheles stephensi, Anopheles darlingi, Aedes aegypti, Aedes albopictus, Culex pipiens quinquefasciatus, and now An. funestus—a pattern is emerging. First, ubiquitous protein families are recruited for a salivary role, such as members of the antigen-5 family and enzymes of nucleotide and carbohydrate catabolism. Second, a group of protein families exclusive to blood-feeding Nematocera includes the abundantly expressed D7 proteins also found in sand flies and Culicoides. A third group of proteins, only found in Culicidae, includes the 30-kDa allergen family and several mucins. Finally, ten protein and peptide families, five of them multigenic, are exclusive to anophelines. Among these proteins may reside good epidemiological markers to measure human exposure to anopheline species such as An. funestus and An. gambiae.
PMCID: PMC1853278  PMID: 17244545
Malaria; Hematophagy; Salivary glands; Vector; Saliva
17.  An annotated catalogue of salivary gland transcripts in the adult female mosquito, Ædes ægypti* 
BMC Genomics  2007;8:6.
Saliva of blood-sucking arthropods contains a cocktail of antihemostatic agents and immunomodulators that help blood feeding. Mosquitoes additionally feed on sugar meals and have specialized regions of their glands containing glycosidases and antimicrobials that might help control bacterial growth in the ingested meals. To expand our knowledge on the salivary cocktail of Ædes ægypti, a vector of dengue and yellow fevers, we analyzed a set of 4,232 expressed sequence tags from cDNA libraries of adult female mosquitoes.
A nonredundant catalogue of 614 transcripts (573 of which are novel) is described, including 136 coding for proteins of a putative secretory nature. Additionally, a two-dimensional gel electrophoresis of salivary gland (SG) homogenates followed by tryptic digestion of selected protein bands and MS/MS analysis revealed the expression of 24 proteins. Analysis of tissue-specific transcription of a subset of these genes revealed at least 31 genes whose expression is specific or enriched in female SG, whereas 24 additional genes were expressed in female SG and in males but not in other female tissues. Most of the 55 proteins coded by these SG transcripts have no known function and represent high-priority candidates for expression and functional analysis as antihemostatic or antimicrobial agents. An unexpected finding is the occurrence of four protein families specific to SG that were probably a product of horizontal transfer from prokaryotic organisms to mosquitoes.
Overall, this paper contributes to the novel identification of 573 new transcripts, or near 3% of the Æ. ægypti proteome assuming a 20,000-protein set, and to the best-described sialome of any blood-feeding insect.
PMCID: PMC1790711  PMID: 17204158
18.  A transcriptome analysis of the Aedes aegypti vitellogenic fat body 
Aedes (Stegomyia) aegypti is an important dengue vector in tropical and subtropical zones throughout the world. A transcriptome of Ae. aegypti vitellogenic fat bodies is described here. The fat body is a dynamic tissue that participates in multiple biochemical functions of intermediate metabolism. A total of 589 randomly selected cDNAs were assembled into 262 clusters based on their primary sequence similarities. The putative translated proteins were classified into categories based on their function in accordance with significant similarity using the BlastX at NCBI FTP site and Pfam (Bateman et al. 2000) and SMART (Schultz et al. 2000) databases. The characterization of transcripts expressed in the fat body of Ae. aegypti at 24 hours post blood meal provides a basic tool for understanding the processes occurring in this organ and could identify putative new genes whose promoters can be used to specifically express transgenes in the fat bodies of Ae. aegypti.
PMCID: PMC2990292  PMID: 19537968

Results 1-18 (18)