All adult mosquitoes take sugar meals, and most adult females also take blood meals to develop eggs. Salivary glands (SG) of males are thus much smaller and do not contain many of the antihemostatic and antiinflammatory compounds found in females. In the past 5 years, transcriptome analyses have identified nearly 70 different genes expressed in adult female SG. For most of these, no function can be assigned in either blood or sugar feeding. Exceptionally, Toxorhynchites mosquitoes are unusual in that they never feed on blood, and the SG of adults are identical in both sexes. Transcriptome analysis of the adult SG of this mosquito was performed to increase knowledge of the evolution of blood feeding—and to identify polypeptide families associated with sugar feeding—in mosquitoes.
Salivary glands; Transcriptome; Mosquito; Hematophagy
Malaria sporozoites must invade the salivary glands of mosquitoes for maturation before transmission to vertebrate hosts. The duration of the sporogonic cycle within the mosquitoes ranges from 10 to 21 days depending on the parasite species and temperature. During blood feeding salivary gland proteins are injected into the vertebrate host, along with malaria sporozoites in the case of an infected mosquito. To identify salivary gland proteins depleted after blood feeding of female Anopheles campestris-like, a potential malaria vector of Plasmodium vivax in Thailand, two-dimensional gel electrophoresis and nano-liquid chromatography-mass spectrometry techniques were used. Results showed that 19 major proteins were significantly depleted in three to four day-old mosquitoes fed on a first blood meal. For the mosquitoes fed the second blood meal on day 14 after the first blood meal, 14 major proteins were significantly decreased in amount. The significantly depleted proteins in both groups included apyrase, 5′-nucleotidase/apyrase, D7, D7-related 1, short form D7r1, gSG6, anti-platelet protein, serine/threonine-protein kinase rio3, putative sil1, cyclophilin A, hypothetical protein Phum_PHUM512530, AGAP007618-PA, and two non-significant hit proteins. To our knowledge, this study presents for the first time the salivary gland proteins that are involved in the second blood feeding on the day corresponding to the transmission period of the sporozoites to new mammalian hosts. This information serves as a basis for future work concerning the possible role of these proteins in the parasite transmission and the physiological processes that occur during the blood feeding.
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.
Mosquitoes are able to adapt to feed on blood by the salivary glands which created a protein that works against the haemostasis process. This study aims to investigate the salivary glands proteins expression of 50 adult female An. dirus A mosquitoes, a main vector of malaria in Thailand, each group with an age of 5 days which were artificial membrane fed on sugar, normal blood, blood infected with P. vivax, and blood infected with P. falciparum. Then mosquito salivary gland proteins were analyzed by SDS-PAGE on days 0, 1, 2, 3, and 4 after feeding. The findings revealed that the major salivary glands proteins had molecular weights of 62, 58, 43, 36, 33, 30, and 18 kDa. One protein band of approximately 13 kDa was found in normal blood and blood infected with P. vivax fed on day 0. A stronger protein band, 65 kDa, was expressed from the salivary glands of mosquitoes fed with P. vivax- or P. falciparum-infected blood on only day 0, but none on days 1 to 4. The study shows that salivary glands proteins expression of An. dirus may affect the malaria parasite life cycle and the ability of mosquitoes to transmit malaria parasites in post-24-hour disappearance observation.
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.
Malaria; Hematophagy; Salivary glands; Vector; Saliva
The Anopheles gambiae salivary gland protein 6 (gSG6) is a small protein specifically found in the salivary glands of adult female mosquitoes. We report here the expression of a recombinant form of the protein and we show that in vivo gSG6 is expressed in distal-lateral lobes and is secreted with the saliva while the female mosquito probes for feeding. Injection of gSG6 dsRNA into adult An. gambiae females results in decreased gSG6 protein levels, increased probing time and reduced blood feeding ability. gSG6 orthologs have been found so far only in the salivary glands of Anopheles stephensi and Anopheles funestus, both members of the Cellia subgenus. We report here the gSG6 sequence from five additional anophelines, four species of the An. gambiae complex and Anopheles freeborni, a member of the subgenus Anopheles. We conclude that gSG6 plays some essential blood feeding role and was recruited in the anopheline subfamily most probably after the separation of the lineage which gave origin to Cellia and Anopheles subgenera.
Anopheles gambiae; gSG6; salivary glands; saliva; blood feeding
Sporozoites are an invasive stage of the malaria parasite in both the mosquito vector and the vertebrate host. We developed an in vivo assay for mosquito salivary gland invasion by preparing Plasmodium gallinaceum sporozoites from infected Aedes aegypti mosquitoes under physiological conditions and inoculating them into uninfected female Ae. aegypti. Sporozoites from mature oocysts were isolated from mosquito abdomens 10 or 11 d after an infective blood meal. Salivary gland sporozoites were isolated 13 or 14 d after an infective blood meal. Purified oocyst sporozoites that were inoculated into uninfected female mosquitoes invaded their salivary glands. Using the same assay system, sporozoites derived from salivary glands did not reinvade the salivary glands after inoculation. Conversely, as few as 10 to 50 salivary gland sporozoites induced infection in chickens, while only 2 of 10 chickens inoculated with 5,000 oocyst sporozoites were infected. Both sporozoite populations were found to express a circumsporozoite protein on the sporozoite surface as determined by immunofluorescence assay and circumsporozoite precipitation test using a circumsporozoite protein-specific monoclonal antibody. We conclude that molecules other than this circumsporozoite protein may be responsible for the differential invasion of mosquito salivary glands or infection of the vertebrate host.
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.
Dendritic cells; T cells; Aedes aegypti; Saliva; Apoptosis
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.
The salivary glands of hematophagous animals contain a complex cocktail that interferes with the host hemostasis and inflammation pathways, thus increasing feeding success. Fleas represent a relatively recent group of insects that evolved hematophagy independently of other insect orders.
Analysis of the salivary transcriptome of the flea Xenopsylla cheopis, the vector of human plague, indicates that gene duplication events have led to a large expansion of a family of acidic phosphatases that are probably inactive, and to the expansion of the FS family of peptides that are unique to fleas. Several other unique polypeptides were also uncovered. Additionally, an apyrase-coding transcript of the CD39 family appears as the candidate for the salivary nucleotide hydrolysing activity in X.cheopis, the first time this family of proteins is found in any arthropod salivary transcriptome.
Analysis of the salivary transcriptome of the flea X. cheopis revealed the unique pathways taken in the evolution of the salivary cocktail of fleas. Gene duplication events appear as an important driving force in the creation of salivary cocktails of blood feeding arthropods, as was observed with ticks and mosquitoes. Only five other flea salivary sequences exist at this time at NCBI, all from the cat flea C. felis. This work accordingly represents the only relatively extensive sialome description of any flea species. Sialotranscriptomes of additional flea genera will reveal the extent that these novel polypeptide families are common throughout the Siphonaptera.
The hosts for Antricola delacruzi ticks are insectivorous, cave-dwelling bats on which only larvae are found. The mouthparts of nymphal and adult A. delacruzi are compatible with scavenging feeding because the hypostome is small and toothless. How a single blood meal of a larva provides energy for several molts as well as for oviposition by females is not known. Adults of A. delacruzi possibly feed upon an unknown food source in bat guano, a substrate on which nymphal and adult stages are always found. Guano produced by insectivorous bats contains twice the amount of protein and 60 times the amount of iron as beef. In addition, bacteria and chitin-rich fungi proliferate on guano. Comparative data on the transcriptome of the salivary glands of A. delacruzi is nonexistent and would help to understand the physiological adaptations of salivary glands that accompany different sources of food as well as the steps taken by the Acari towards haematophagy, believed to have evolved from scavenging dead animals. Annotation of the transcriptome of salivary glands from female instars of A. delacruzi collected on guano categorized 5.7% of the clusters of expressed genes as putative secreted proteins. They included abundantly expressed TIL domain-containing proteins (possible anti-microbials), an abundantly expressed protein similar to a serum amyloid found in the sialotranscriptomes of Ornithodoros spp., a savignygrin, a family of mucin/peritrophin/cuticle-like proteins, antimicrobials and an HIV envelope-like glycoprotein also found in soft ticks. When comparing the transcriptome of A. delacruzi with those of blood-feeding female soft and hard ticks some notable differences were observed; they consisted of the following transcripts over- or under-represented or absent in the sialotranscriptome of A. delacuzi that may reflect its source of food: ferritin, mucins with chitin-binding domains and TIL domain-containing proteins versus lipocalins, basic tail proteins, metalloproteases, glycine-rich proteins and Kunitz protease inhibitors, respectively.
Antricola delacruzi; Hematophagy; Scavenging; Transcriptome; Salivary glands; Bat guano
Saliva of Aedes aegypti contains a complex array of proteins essential for both blood feeding and pathogen transmission. A large numbers of those proteins are classified as unknown in regard to their function(s). Understanding the dynamic interactions at the mosquito-host interface can be achieved in part by characterizing mosquito salivary gland gene expression relative to blood feeding. Towards this end, we developed an oligonucleotide microarray representing 463 transcripts to determine differential regulation of salivary gland genes. This microarray was used to investigate the temporal gene expression pattern of Ae. aegypti salivary gland transcriptome at different times post-blood feeding. Expression of the majority of salivary gland genes (77–87%) did not change significantly as a result of blood feeding, while 8 to 20% of genes were down-regulated and 2.8 to 11.6% genes were up-regulated. Up-regulated genes included defensins, mucins and other immune related proteins. Odorant-binding protein was significantly down-regulated. Among unknown function proteins, several were up-regulated during the first three hours post-blood feeding and one was significantly down-regulated. Quantitative real-time RT-PCR was used to substantiate differential expression patterns of five randomly selected genes. Linear regression analysis revealed a high degree of correlation (R2 > 0.89) between oligonucleotide microarray and quantitative RT-PCR data. To our knowledge, this is the first study to investigate differential expression of the Ae. aegypti salivary gland transcriptome upon blood feeding. A microarray provides a robust, sensitive way to investigate differential regulation of mosquito salivary gland genes.
Dipetalogaster maximais 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. Supplemental files can be downloaded from http://exon.niaid.nih.gov/transcriptome/D_maxima/Dm-S1-web.xls and http://exon.niaid.nih.gov/transcriptome/D_maxima/Dm-S2-web.xls.
Saliva; hematophagy; transcriptome; proteome; triatomine; D. maxima
Aedes aegypti mosquitoes are the main vectors of dengue viruses to humans. Understanding their biology and interactions with the pathogen are prerequisites for development of dengue transmission control strategies. Mosquito salivary glands are organs involved directly in pathogen transmission to vertebrate hosts. Information on the spatial distribution of gene expression in these organs is expected to assist in the development of novel disease control strategies, including those that entail the release of transgenic mosquitoes with impaired vector competence.
We report here the hybridization in situ patterns of 30 transcripts expressed in the salivary glands of adult Ae. aegypti females. Distinct spatial accumulation patterns were identified. The products of twelve genes are localized exclusively in the proximal-lateral lobes. Among these, three accumulate preferentially in the most anterior portion of the proximal-lateral lobe. This pattern revealed a salivary gland cell type previously undescribed in Ae. aegypti, which was validated by transmission electron microscopy. Five distinct gene products accumulate in the distal-lateral lobes and another five localize in the medial lobe. Seven transcripts are found in the distal-lateral and medial lobes. The transcriptional product of one gene accumulates in proximal- and distal-lateral lobes. Seven genes analyzed by quantitative PCR are expressed constitutively. The most abundant salivary gland transcripts are those localized within the proximal-lateral lobes, while previous work has shown that the distal-lateral lobes are the most active in protein synthesis. This incongruity suggests a role for translational regulation in mosquito saliva production.
Transgenic mosquitoes with reduced vector competence have been proposed as tools for the control of dengue virus transmission. Expression of anti-dengue effector molecules in the distal-lateral lobes of Ae. aegypti salivary glands has been shown to reduce prevalence and mean intensities of viral infection. We anticipate greater efficiency of viral suppression if effector genes are expressed in all lobes of the salivary glands. Based on our data, a minimum of two promoters is necessary to drive the expression of one or more anti-dengue genes in all cells of the female salivary glands.
Ticks are serious haematophagus arthropod pests and are only second to mosquitoes as vectors of diseases of humans and animals. The salivary glands of the slower feeding hard ticks such as Haemaphysalis longicornis are a rich source of bioactive molecules and are critical to their biologic success, yet distinct molecules that help prolong parasitism on robust mammalian hosts and achieve blood-meals remain unidentified. Here, we report on the molecular and biochemical features and precise functions of a novel Kunitz inhibitor from H. longicornis salivary glands, termed Haemangin, in the modulation of angiogenesis and in persistent blood-feeding. Haemangin was shown to disrupt angiogenesis and wound healing via inhibition of vascular endothelial cell proliferation and induction of apoptosis. Further, this compound potently inactivated trypsin, chymotrypsin, and plasmin, indicating its antiproteolytic potential on angiogenic cascades. Analysis of Haemangin-specific gene expression kinetics at different blood-feeding stages of adult ticks revealed a dramatic up-regulation prior to complete feeding, which appears to be functionally linked to the acquisition of blood-meals. Notably, disruption of Haemangin-specific mRNA by a reverse genetic tool significantly diminished engorgement of adult H. longicornis, while the knock-down ticks failed to impair angiogenesis in vivo. To our knowledge, we have provided the first insights into transcriptional responses of human microvascular endothelial cells to Haemangin. DNA microarray data revealed that Haemangin altered the expression of 3,267 genes, including those of angiogenic significance, further substantiating the antiangiogenic function of Haemangin. We establish the vital roles of Haemangin in the hard tick blood-feeding process. Moreover, our results provide novel insights into the blood-feeding strategies that enable hard ticks to persistently feed and ensure full blood-meals through the modulation of angiogenesis and wound healing processes.
Ticks are notorious ectoparasites that exclusively feed on a host's blood for a period of 10 days or longer. Upon blood-feeding, an adult female tick gains 100–200 times its body weight compared to its pre-feeding stage. Despite the host's armoury of rejection mechanisms, ticks manage to remain attached until a full blood-meal is ensured. The molecular machineries that make the tick a success with its feeding, however, remain unknown. We demonstrate that the Kunitz-like protein Haemangin, identified from the salivary glands of the tick Haemaphysalis longicornis, plays vital roles in blood-feeding success. Using both cell- and chick embryo–based bioassays, we have shown that Haemangin efficiently disrupted angiogenesis and wound healing processes, enabling ticks to remain attached and allowing persistent feeding. Additionally, in a rabbit model, we reveal that an elevated expression of Haemangin is associated with the acquisition of full blood-meals. Importantly, Haemangin-knockdown ticks fail to prevent angiogenesis in the host's tissues and consequently achieve only a poor blood-meal as compared to normal ticks. We conclude that Haemangin is vital for ticks' survival and can be a novel therapeutic target against ticks and tick-borne diseases, including tumor angiogenesis.
Saliva of blood sucking arthropods contains compounds that antagonize their hosts' hemostasis, which include platelet aggregation, vasoconstriction and blood clotting; saliva of these organisms also has anti-inflammatory and immunomodullatory properties. Perhaps because hosts mount an active immune response against these compounds, the diversity of these compounds is large even among related blood sucking species. Because of these properties, saliva helps blood feeding as well as help the establishment of pathogens that can be transmitted during blood feeding.
We have obtained 1,626,969 reads by pyrosequencing a salivary gland cDNA library from adult females Amblyomma maculatum ticks at different times of feeding. Assembly of this data produced 72,441 sequences larger than 149 nucleotides from which 15,914 coding sequences were extracted. Of these, 5,353 had >75% coverage to their best match in the non-redundant database from the National Center for Biotechnology information, allowing for the deposition of 4,850 sequences to GenBank. The annotated data sets are available as hyperlinked spreadsheets. Putative secreted proteins were classified in 133 families, most of which have no known function.
This data set of proteins constitutes a mining platform for novel pharmacologically active proteins and for uncovering vaccine targets against A. maculatum and the diseases they carry.
The saliva of hematophagous arthropods contains potent anti-inflammatory and antihemostatic activities that promote acquisition of the blood meal and enhance infection with pathogens. We have shown that polymorphonuclear leukocytes (PMN) treated with the saliva of the tick Ixodes scapularis have reduced expression of β2 integrins, impaired PMN adherence, and reduced killing of Borrelia burgdorferi, the causative agent of Lyme disease. Here we describe two Ixodes proteins that are induced upon tick feeding and expressed predominantly in the salivary glands. Using saliva harvested from ticks with reduced levels of ISL 929 and ISL 1373 through targeted RNA interference knockdown, as well as purified recombinant proteins, we show the effects of these proteins on downregulation of PMN integrins and inhibition of the production of O2− by PMN in vitro. Mice immunized with ISL 929/1373 had increased numbers of PMN at the site of tick attachment and a lower spirochete burden in the skin and joints 21 days after infection compared to control-immunized animals. Our results suggest that ISL 929 and ISL 1373 contribute to the inhibition of PMN functions shown previously with tick saliva and support important roles for these inhibitory proteins in the modulation of PMN function in vivo.
Anopheles funestus is a principal vector of malaria across much of tropical Africa and is considered one of the most efficient of its kind, yet studies of this species have lagged behind those of its broadly sympatric congener, An. gambiae. In aid of future genomic sequencing of An. funestus, we explored the whole body transcriptome, derived from mixed stage progeny of wild-caught females from Mali, West Africa.
Here we report the functional annotation and comparative genomics of 2,005 expressed sequence tags (ESTs) from An. funestus, which were assembled with a previous EST set from adult female salivary glands from the same mosquito. The assembled ESTs provided for a nonredundant catalog of 1,035 transcripts excluding mitochondrial sequences.
Comparison of the An. funestus and An. gambiae transcriptomes using computational and macroarray approaches revealed a high degree of sequence identity despite an estimated 20–80 MY divergence time between lineages. A phylogenetically broader comparative genomic analysis indicated that the most rapidly evolving proteins– those involved in immunity, hematophagy, formation of extracellular structures, and hypothetical conserved proteins– are those that probably play important roles in how mosquitoes adapt to their nutritional and external environments, and therefore could be of greatest interest in disease control.
Ixodid ticks are notorious blood-sucking ectoparasites and are completely dependent on blood-meals from hosts. In addition to the direct severe effects on health and productivity, ixodid ticks transmit various deadly diseases to humans and animals. Unlike rapidly feeding vessel-feeder hematophagous insects, the hard ticks feed on hosts for a long time (5−10 days or more), making a large blood pool beneath the skin. Tick's salivary glands produce a vast array of bio-molecules that modulate their complex and persistent feeding processes. However, the specific molecule that functions in the development and maintenance of a blood pool is yet to be identified. Recently, we have reported on longistatin, a 17.8-kDa protein with two functional EF-hand Ca++-binding domains, from the salivary glands of the disease vector, Haemaphysalis longicornis, that has been shown to be linked to blood-feeding processes. Here, we show that longistatin plays vital roles in the formation of a blood pool and in the acquisition of blood-meals. Data clearly revealed that post-transcriptional silencing of the longistatin-specific gene disrupted ticks' unique ability to create a blood pool, and they consequently failed to feed and replete on blood-meals from hosts. Longistatin completely hydrolyzed α, β and γ chains of fibrinogen and delayed fibrin clot formation. Longistatin was able to bind with fibrin meshwork, and activated fibrin clot-bound plasminogen into its active form plasmin, as comparable to that of tissue-type plasminogen activator (t-PA), and induced lysis of fibrin clot and platelet-rich thrombi. Plasminogen activation potentiality of longistatin was increased up to 4 times by soluble fibrin. Taken together, our results suggest that longistatin may exert potent functions both as a plasminogen activator and as an anticoagulant in the complex scenario of blood pool formation; the latter is critical to the feeding success and survival of ixodid ticks.
Ixodid ticks are serious blood-sucking ectoparasites that are essentially dependent on blood-meals from hosts for survival. The feeding mechanism of hard ticks, however, is very complex and is quite different from that of blood-sucking insects that suck blood rapidly and directly from blood vessels. Hard ticks suck blood for quite a long period (5−10 days or more) by making a large blood pool beneath the skin. Despite the fact that mammalian hosts are armored with strong blood clotting machineries, ticks manage to keep the blood in a fluid state and to maintain a blood pool until a full blood-meal is secured. However, very little is known about the anti-hemostatic mechanisms by which ticks cleverly manipulate the host's blood clotting cascade and make blood-meals available. Here, we show that longistatin, a salivary gland protein identified from the tick Haemaphysalis longicornis, can efficiently manipulate the host's blood clotting machineries, such as fibrinogen, fibrin and plasminogen, and can help prevent cascade of blood clotting. In conclusion, longistatin plays a crucial role in the blood-feeding success of hard ticks and can be a novel therapeutic target against ticks and tick-borne diseases, including human occlusive cardiovascular diseases like thrombosis.
The saliva of blood sucking insects contains potent pharmacologically active components that assist them in counteracting the host hemostatic and inflammatory systems during blood feeding. In addition, sand fly salivary proteins affect host immunity and have the potential to be a vaccine against Leishmania infection. In the present study, the salivary gland transcripts of Lutzomyia ayacuchensis, a vector of cutaneous leishmaniasis in Ecuadorian and Peruvian Andes, were analyzed by sequencing randomly selected clones of the salivary gland cDNA library of this sand fly. This resulted in the identification of the most abundant transcripts coding for secreted proteins. These proteins were homologous to the salivary molecules present in other sand flies including the RGD-containing peptide, PpSP15/SL1 family protein, yellow-related protein, putative apyrase, antigen 5-related protein, D7 family protein, and 27 kDa salivary protein. Of note, homologues of maxadilan, an active vasodilator abundantly present in saliva of Lutzomyia longipalpis, were not identified. This analysis is the first description of salivary proteins from a sand fly of the subgenus Helcocyrtomyia and from vector of cutaneous leishmaniasis in the New World. The present analysis will provide further insights into the evolution of salivary components in blood sucking arthropods.
Lutzomyia ayacuchensis; Salivary gland; Transcript; Bioinformatics; cDNA library
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.
mosquito; salivary gland; sialome; transcriptome; La Crosse virus
When attempting to feed on their hosts, ticks face the problem of host hemostasis (the vertebrate mechanisms that prevent blood loss), inflammation (that can produce itching or pain and thus initiate defensive behavior on their hosts) and immunity (by way of both cellular and humoral responses). Against these barriers, ticks evolved a complex and sophisticated pharmacological armamentarium, consisting of bioactive lipids and proteins, to assist blood feeding. Recent progress in transcriptome research has uncovered that hard ticks have hundreds of different proteins expressed in their salivary glands, the majority of which have no known function, and include many novel protein families (e.g., their primary structure is unique to ticks). This review will address the vertebrate mechanisms of these barriers as a guide to identify the possible targets of these large numbers of known salivary proteins with unknown function. We additionally provide a supplemental table that catalogues over 3,500 putative salivary proteins from various tick species, which might assist the scientific community in the process of functional identification of these unique proteins. This supplemental file is accessble from http://exon.niaid.nih.gov/transcriptome/tick_review/Sup-Table-1.xls.gz.
Blood-sucking arthropods salivary glands (SGs) contain a remarkable diversity of antihemostatics. The aim of this study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades.
Methods and Results
Several L. longipalpis salivary proteins were expressed in HEK293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, non-competitive, and reversible inhibitor of Factor Xa (FXa). Notably, Lufaxin primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, DEGR- FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both PT and aPTT. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with a KD ~3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents PAR2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs aPTT ex vivo, implying that it works as an anticoagulant in vivo. Finally, SG of sandflies was found to inhibit FXa and to interact with the enzyme.
Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and antiinflamatory activities. It is a useful tool to understand FXa structural features and its role in pro-hemostatic and pro-inflammatory events.
hematophagy; thrombosis; leishmaniasis; vector biology; microcirculation
Controlled sex-, stage- and tissue-specific expression of anti-pathogen effector molecules is important for genetic engineering strategies to control mosquito-borne diseases. Adult female salivary glands are involved in pathogen transmission to human hosts and are target sites for expression of anti-pathogen effector molecules. The Aedes aegypti 30K a and 30K b genes are expressed exclusively in adult female salivary glands and are transcribed divergently from start sites separated by 263 nucleotides. The intergenic, 5’- and 3’-end untranslated regions of both genes are sufficient to express simultaneously two different transgene products in the distal-lateral lobes of the female salivary glands. An anti-dengue effector gene, Mnp, driven by the 30K b promoter, expresses an inverted-repeat RNA with sequences derived from the premembrane protein-encoding region of the dengue virus serotype 2 genome and reduces significantly the prevalence and mean intensities of viral infection in mosquito salivary glands and saliva.
Dengue; mosquito; salivary glands; promoter; transgenesis; Aedes aegypti
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.