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1.  An Insight into the Sialotranscriptome of Simulium nigrimanum, a Black Fly Associated with Fogo Selvagem in South America 
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.
PMCID: PMC2877412  PMID: 20519601
2.  An Insight into the Sialome of the Black Fly, Simulium vittatum 
Journal of proteome research  2009;8(3):1474-1488.
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 in 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 and
PMCID: PMC2778207  PMID: 19166301
Simulium vittatum; black fly; sialotranscriptomes; salivary gland transcriptome; sialome; proteome; hematophagy; onchocerciasis
3.  Simukunin from the Salivary Glands of the Black Fly Simulium vittatum Inhibits Enzymes That Regulate Clotting and Inflammatory Responses 
PLoS ONE  2012;7(2):e29964.
Black flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized.
Methodology/Principal Findings
Two Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins are inhibitors of serine proteases, we hypothesized that SV-66 and/or −170 were involved in the anticoagulant activity of black fly saliva. Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation. Mutational analysis suggested that SV-66 is a canonical BPTI-like inhibitor. Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin. Surface plasmon resonance studies indicated that rSV-66 bound with highest affinity to elastase (KD = 0.4 nM) and to the active site of FXa (KD = 3.07 nM). We propose the name “Simukunin” for this novel protein.
We conclude that Simukunin preferentially inhibits Factor Xa. The inhibition of elastase and cathepsin G further suggests this protein may modulate inflammation, which could potentially affect pathogen transmission.
PMCID: PMC3285612  PMID: 22383955
4.  An insight into the sialome of the frog biting fly, Corethrella appendiculata 
Insect biochemistry and molecular biology  2013;S0965-1748(13)00191-4 10.1016/j.ibmb.2013.10.006.
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. Bloodfeeding 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 bloodfeeding, and novel protein families unique to this species of frog-feeding fly. Bacterial (Wolbachia) and novel viral sequences were also discovered.
PMCID: PMC4035455  PMID: 24514880
Evolution; Culicomorpha; Corethrellidae; Salivary gland; Hematophagy; Medical entomology
5.  An Insight into the Sialotranscriptome of the Cat Flea, Ctenocephalides felis 
PLoS ONE  2012;7(9):e44612.
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.
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.
PMCID: PMC3458046  PMID: 23049752
6.  Identification of Human Semiochemicals Attractive to the Major Vectors of Onchocerciasis 
Entomological indicators are considered key metrics to document the interruption of transmission of Onchocerca volvulus, the etiological agent of human onchocerciasis. Human landing collection is the standard employed for collection of the vectors for this parasite. Recent studies reported the development of traps that have the potential for replacing humans for surveillance of O. volvulus in the vector population. However, the key chemical components of human odor that are attractive to vector black flies have not been identified.
Methodology/Principal Findings
Human sweat compounds were analyzed using GC-MS analysis and compounds common to three individuals identified. These common compounds, with others previously identified as attractive to other hematophagous arthropods were evaluated for their ability to stimulate and attract the major onchocerciasis vectors in Africa (Simulium damnosum sensu lato) and Latin America (Simulium ochraceum s. l.) using electroantennography and a Y tube binary choice assay. Medium chain length carboxylic acids and aldehydes were neurostimulatory for S. damnosum s.l. while S. ochraceum s.l. was stimulated by short chain aliphatic alcohols and aldehydes. Both species were attracted to ammonium bicarbonate and acetophenone. The compounds were shown to be attractive to the relevant vector species in field studies, when incorporated into a formulation that permitted a continuous release of the compound over time and used in concert with previously developed trap platforms.
The identification of compounds attractive to the major vectors of O. volvulus will permit the development of optimized traps. Such traps may replace the use of human vector collectors for monitoring the effectiveness of onchocerciasis elimination programs and could find use as a contributing component in an integrated vector control/drug program aimed at eliminating river blindness in Africa.
Author Summary
Human landing collections, which are the current standard for collecting the black fly vectors of Onchocerca volvulus, the causative agent of river blindness, are inefficient and pose certain ethical issues. As entomological methods are among the primary techniques recommended by the international community for verifying the elimination of onchocerciasis, there is a need to develop alternative methods to collect these vectors. Recent studies have demonstrated that traps baited with CO2 and dirty clothing have the potential to replace human landing collections for this purpose. However, for these traps to be widely applied, it will be necessary to develop a consistent bait formulation. To this end, volatile compounds from human sweat that attract the principal black fly vectors of O. volvulus in Africa and the Americas have been identified and used to optimize traps that specifically collect these insects. To achieve this milestone, we report the use of electroantennography and behavioral assays to identify human compounds that are neurostimulatory to these vectors, and demonstrate that these compounds are attractive to the vectors in field studies using previously developed trap platforms. The development of such a defined bait formulation will permit the widespread use of these traps by onchocerciasis elimination programs in Africa and the Americas.
PMCID: PMC4287528  PMID: 25569240
7.  An insight into the sialome of Glossina morsitans morsitans 
BMC Genomics  2010;11:213.
Blood feeding evolved independently in worms, arthropods and mammals. Among the adaptations to this peculiar diet, these animals developed an armament of salivary molecules that disarm their host's anti-bleeding defenses (hemostasis), inflammatory and immune reactions. Recent sialotranscriptome analyses (from the Greek sialo = saliva) of blood feeding insects and ticks have revealed that the saliva contains hundreds of polypeptides, many unique to their genus or family. Adult tsetse flies feed exclusively on vertebrate blood and are important vectors of human and animal diseases. Thus far, only limited information exists regarding the Glossina sialome, or any other fly belonging to the Hippoboscidae.
As part of the effort to sequence the genome of Glossina morsitans morsitans, several organ specific, high quality normalized cDNA libraries have been constructed, from which over 20,000 ESTs from an adult salivary gland library were sequenced. These ESTs have been assembled using previously described ESTs from the fat body and midgut libraries of the same fly, thus totaling 62,251 ESTs, which have been assembled into 16,743 clusters (8,506 of which had one or more EST from the salivary gland library). Coding sequences were obtained for 2,509 novel proteins, 1,792 of which had at least one EST expressed in the salivary glands. Despite library normalization, 59 transcripts were overrepresented in the salivary library indicating high levels of expression. This work presents a detailed analysis of the salivary protein families identified. Protein expression was confirmed by 2D gel electrophoresis, enzymatic digestion and mass spectrometry. Concurrently, an initial attempt to determine the immunogenic properties of selected salivary proteins was undertaken.
The sialome of G. m. morsitans contains over 250 proteins that are possibly associated with blood feeding. This set includes alleles of previously described gene products, reveals new evidence that several salivary proteins are multigenic and identifies at least seven new polypeptide families unique to Glossina. Most of these proteins have no known function and thus, provide a discovery platform for the identification of novel pharmacologically active compounds, innovative vector-based vaccine targets, and immunological markers of vector exposure.
PMCID: PMC2853526  PMID: 20353571
8.  An insight into the sialome of the soft tick, Ornithodorus parkeri 
While hard ticks (Ixodidae) take several days to feed on their hosts, soft ticks (Argasidae) feed faster, usually taking less than one hour per meal. Saliva assists in the feeding process by providing a cocktail of anti-hemostatic, anti-inflammatory and immunomodullatory compounds. Saliva of hard ticks has been shown to contain several families of genes each having multiple members, while those of soft ticks are relatively unexplored.
Analysis of the salivary transcriptome of the soft tick Ornithodorus parkeri, the vector of the relapsing fever agent Borrelia parkeri, indicates that gene duplication events have led to a large expansion of the lipocalin family, as well as of several genes containing Kunitz domains indicative of serine protease inhibitors, and several other gene families also found in hard ticks. Novel protein families with sequence homology to insulin growth factor-binding protein (prostacyclin-stimulating factor), adrenomedulin, serum amyloid A protein precursor and similar to HIV envelope protein were also characterized for the first time in the salivary gland of a blood-sucking arthropod.
The sialotranscriptome of O. parkeri confirms that gene duplication events are an important driving force in the creation of salivary cocktails of blood-feeding arthropods, as was observed with hard ticks and mosquitoes. Most of the genes coding for expanded families are homologous to those found in hard ticks, indicating a strong common evolutionary path between the two families. As happens to all genera of blood-sucking arthropods, several new proteins were also found, indicating the process of adaptation to blood feeding still continues to recent times.
PMCID: PMC2233652  PMID: 18070662
Ornithodorus parkeri; Ixodidae; Argasidae; Sialotranscriptomes; salivary gland transcriptome; sialome; Tick salivary glands
9.  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
10.  Individual exposure to Simulium bites and intensity of Onchocerca volvulus infection 
Parasites & Vectors  2010;3:53.
Onchocerca volvulus, the causative agent of river blindness, is transmitted through the black fly Simulium damnosum s.l., which breeds in turbulent river waters. To date, the number of flies attacking humans has only been determined by standard fly collectors near the river or the village. In our study, we counted the actual number of attacking and successfully feeding S. damnosum s.l. flies landing on individual villagers during their routine day-time activities in two villages of the Sudan-savannah and rainforest of Cameroon. We compared these numbers to the number of flies caught by a standard vector-collector, one positioned near the particular villager during his/her daily activity and the other sitting at the nearest Simulium breeding site.
Using these data obtained by the two vector-collectors, we were able to calculate the Actual Index of Exposure (AIE). While the AIE in the savannah was on average 6,3%, it was 34% in the rainforest. The Effective Annual Transmission Potential (EATP) for individual villagers was about 20 fold higher in the rainforest compared to the savannah.
Here we show for the first time that it is possible to determine the EATP. Further studies with more subjects are needed in the future. These data are important for the development of future treatment strategies.
PMCID: PMC2910011  PMID: 20565835
11.  The sialotranscriptome of Amblyomma triste, Amblyomma parvum and Amblyomma cajennense ticks, uncovered by 454-based RNA-seq 
Parasites & Vectors  2014;7:430.
Tick salivary constituents antagonize inflammatory, immune and hemostatic host responses, favoring tick blood feeding and the establishment of tick-borne pathogens in hosts during hematophagy. Amblyomma triste, A. cajennense and A. parvum ticks are very important in veterinary and human health because they are vectors of the etiological agents for several diseases. Insights into the tick salivary components involved in blood feeding are essential to understanding vector-pathogen-host interactions, and transcriptional profiling of salivary glands is a powerful tool to do so. Here, we functionally annotated the sialotranscriptomes of these three Amblyomma species, which allowed comparisons between these and other hematophagous arthropod species.
mRNA from the salivary glands of A. triste, A. cajennense and A. parvum ticks fed on different host species were pyrosequenced on a 454-Roche platform to generate four A. triste (nymphs fed on guinea pigs and females fed on dogs) libraries, one A. cajennense (females fed on rabbits) library and one was A. parvum (females fed on dogs) library. Bioinformatic analyses used in-house programs with a customized pipeline employing standard assembly and alignment algorithms, protein databases and protein servers.
Each library yielded an average of 100,000 reads, which were assembled to obtain contigs of coding sequences (CDSs). The sialotranscriptome analyses of A. triste, A. cajennense and A. parvum ticks produced 11,240, 4,604 and 3,796 CDSs, respectively. These CDSs were classified into over 100 distinct protein families with a wide range of putative functions involved in physiological and blood feeding processes and were catalogued in annotated, hyperlinked spreadsheets. We highlighted the putative transcripts encoding saliva components with critical roles during parasitism, such as anticoagulants, immunosuppressants and anti-inflammatory molecules. The salivary content underwent changes in the abundance and repertoire of many transcripts, which depended on the tick and host species.
The annotated sialotranscriptomes described herein richly expand the biological knowledge of these three Amblyomma species. These comprehensive databases will be useful for the characterization of salivary proteins and can be applied to control ticks and tick-borne diseases.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-3305-7-430) contains supplementary material, which is available to authorized users.
PMCID: PMC4261526  PMID: 25201527
Ticks; Amblyomma cajennense; Amblyomma parvum; Amblyomma triste; Salivary gland; Saliva; Transcriptome; RNA-seq
12.  Elimination of Onchocerciasis from Mexico 
PLoS Neglected Tropical Diseases  2015;9(7):e0003922.
Mexico is one of the six countries formerly endemic for onchocerciasis in Latin America. Transmission has been interrupted in the three endemic foci of that country and mass drug distribution has ceased. Three years after mass drug distribution ended, post-treatment surveillance (PTS) surveys were undertaken which employed entomological indicators to check for transmission recrudescence.
Methodology/Principal findings
In-depth entomologic assessments were performed in 18 communities in the three endemic foci of Mexico. None of the 108,212 Simulium ochraceum s.l. collected from the three foci were found to contain parasite DNA when tested by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), resulting in a maximum upper bound of the 95% confidence interval (95%-ULCI) of the infective rate in the vectors of 0.035/2,000 flies examined. This is an order of magnitude below the threshold of a 95%-ULCI of less than one infective fly per 2,000 flies tested, the current entomological criterion for interruption of transmission developed by the international community. The point estimate of seasonal transmission potential (STP) was zero, and the upper bound of the 95% confidence interval for the STP ranged from 1.2 to 1.7 L3/person/season in the different foci. This value is below all previous estimates for the minimum transmission potential required to maintain the parasite population.
The results from the in-depth entomological post treatment surveillance surveys strongly suggest that transmission has not resumed in the three foci of Mexico during the three years since the last distribution of ivermectin occurred; it was concluded that transmission remains undetectable without intervention, and Onchocerca volvulus has been eliminated from Mexico.
Author Summary
Onchocerciasis, or river blindness, is one of the neglected tropical diseases targeted by the international community for elimination. In Mexico, onchocerciasis was historically endemic in three foci, which included Northern Chiapias, Southern Chiapas and Oaxaca. Both the criteria for verification of elimination and for post-treatment surveillance developed by the international community rely heavily on the use of entomological metrics. The absence of evidence of ongoing transmission of the parasite three years after mass drug distribution has been halted is considered to be evidence that elimination efforts have been successful. In the present study, we report entomological assessments carried out in the three endemic foci in Mexico that were performed three years following the end of mass drug distribution in each focus. None of the over 100,000 Simulium ochraceum s.l. vector black flies collected from sentinel and extra-sentinel communities in these foci were found to contain parasite DNA, suggesting vector parasite contact was non-existent. This data suggest that elimination of onchocerciasis from Mexico has been achieved.
PMCID: PMC4498594  PMID: 26161558
13.  The mermithid species Isomermis lairdi (Nematoda, Mermithidae), previously only known in Africa, found in Europe 
ZooKeys  2014;1-11.
The present work contributs to the knowledge on the aquatic mermithids (Nematoda, Mermithidae) occurring in black flies – an insufficiently studied group of parasitic nematodes. Isomermis lairdi Mondet, Poinar & Bernadou, 1977, described from larvae of Simulium damnosum Theobald, 1903 in Western Africa, is reported to occur in Bulgaria. The species was isolated from larvae of Simulium ornatum Meigen, 1818 in a local population of simuliids in a mountain stream near Jeleznitsa Village, Sofia district. Postparasitic juveniles of mermithids were obtained from the hosts and reared to the adult stage. The species was identified by morphological and morphometrical characters of postparasitic juveniles, and of male and female individuals. In the summer of 2012 a relatively high rate of mermithid infection in a local host population was detected (prevalence up to 44.1%). In August of the next year host abundance had considerably declined and other simuliid species, Simulium variegatum Meigen, 1818 and Simulium reptans (Linnaeus, 1758), predominated in the investigated locality. In West Africa, Isomermis lairdi is considered to be a potential biological agent for reducing the population density of the Simulium damnosum complex – the main vector of human onchocerciasis. In Europe, species of the Simulium ornatum complex are among the vectors of onchocerciasis of cattle and deer. The mermithids presumably play a certain role in the epidemiology of these diseases. A brief discussion on the taxonomy of the genus Isomermis Coman, 1953, and of the feasibility of molecular methods in mermithid taxonomy is provided. The species Isomermis lairdi is reported for the first time from Europe.
PMCID: PMC4258736  PMID: 25493063
Entomoparasitic nematodes; morphology; taxonomy; parasite ecology; distribution; Simulium ornatum; disease vectors; black fly control; Bulgaria
14.  A further insight into the sialome of the tropical bont tick, Amblyomma variegatum 
BMC Genomics  2011;12:136.
Ticks--vectors of medical and veterinary importance--are themselves also significant pests. Tick salivary proteins are the result of adaptation to blood feeding and contain inhibitors of blood clotting, platelet aggregation, and angiogenesis, as well as vasodilators and immunomodulators. A previous analysis of the sialotranscriptome (from the Greek sialo, saliva) of Amblyomma variegatum is revisited in light of recent advances in tick sialomes and provides a database to perform a proteomic study.
The clusterized data set has been expertly curated in light of recent reviews on tick salivary proteins, identifying many new families of tick-exclusive proteins. A proteome study using salivary gland homogenates identified 19 putative secreted proteins within a total of 211 matches.
The annotated sialome of A. variegatum allows its comparison to other tick sialomes, helping to consolidate an emerging pattern in the salivary composition of metastriate ticks; novel protein families were also identified. Because most of these proteins have no known function, the task of functional analysis of these proteins and the discovery of novel pharmacologically active compounds becomes possible.
PMCID: PMC3060141  PMID: 21362191
15.  An insight into the sialome of the oriental rat flea, Xenopsylla cheopis (Rots) 
BMC Genomics  2007;8:102.
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.
PMCID: PMC1876217  PMID: 17437641
16.  Identification of Communal Oviposition Pheromones from the Black Fly Simulium vittatum 
PLoS ONE  2015;10(3):e0118904.
The suite of pheromones that promote communal oviposition by Simulium vittatum, a North American black fly species, was identified and characterized using gas chromatography-mass spectrometry, electrophysiological, and behavioral bioassays. Behavioral assays demonstrated that communal oviposition was induced by egg-derived compounds that were active at short range and whose effect was enhanced through direct contact. Three compounds (cis-9-tetradecen-1-ol, 1-pentadecene, and 1-tridecene) were identified in a non-polar solvent extract of freshly deposited S. vittatum eggs that were capable of inducing the oviposition response. Electroantennography demonstrated that two of these three compounds (1-pentadecene and 1-tridecene) actively stimulated antennal neurons. Identification of the oviposition pheromones of this family may be helpful in developing control measures for nuisance black flies and for medically-important species such as Simulium damnosum sensu lato.
PMCID: PMC4364695  PMID: 25786206
17.  Evaluation of a Community-Based Trapping Program to Collect Simulium ochraceum sensu lato for Verification of Onchocerciasis Elimination 
Collection of the black fly vectors of onchocerciasis worldwide relies upon human landing collections. Recent studies have suggested that the Esperanza Window Trap baited with a human scent lure and CO2 had the potential to replace human hosts for the collection of Simulium ochraceum sensu lato in Southern Chiapas focus, Mexico. The feasibility of utilizing these traps in a community-based approach for the collection of S. ochraceum s.l. was evaluated.
Methodology/Principal findings
Local residents of a formerly endemic extra-sentinel community for onchocerciasis were trained to carry out collections using the traps. The residents operated the traps over a 60-day period and conducted parallel landing collections, resulting in a total of 28,397 vector black flies collected. None of the flies collected were found to contain parasite DNA when tested by a polymerase chain reaction assay targeting a parasite specific sequence, resulting in a point estimate of infection in the vectors of zero, with an upper bound of the 95% confidence interval 0.13 per 2,000. This meets the accepted criterion for demonstrating an interruption of parasite transmission.
These data demonstrate that Esperanza Window Traps may be effectively operated by minimally trained residents of formerly endemic communities, resulting in the collection of sufficient numbers of flies to verify transmission interruption of onchocerciasis. The traps represent a viable alternative to using humans as hosts for the collection of vector flies as part of the verification of onchocerciasis elimination.
Author Summary
Onchocerciasis, or river blindness, is a neglected tropical disease that has been identified by the international community as a candidate for elimination. Both the criteria for verification of elimination and for post-treatment surveillance developed by the international community rely heavily on the use of entomological metrics. Large numbers of vector black flies must be collected to satisfy these metrics. The current standard method for collection of vector black flies for this purpose is human landing collections, is both inefficient and potentially hazardous to the collectors. Here, we report studies evaluating a community-based trial of an inexpensive trap made largely from locally available materials for the replacement of fly collection teams. Traps were provided to residents of a formerly onchocerciasis endemic community in Mexico, and the residents allowed to operate the traps over a 60 day period. The number of flies collected was sufficient to meet the current international criteria necessary to verify that the community was free of O. volvulus transmission. These findings suggest that community based operation of this simple trap might replace human landing collections in the process of verifying the interruption of transmission of onchocerciasis.
PMCID: PMC4207651  PMID: 25340517
18.  Purification and characterization of a novel defensin from the salivary glands of the black fly, Simulium bannaense 
Parasites & Vectors  2015;8:71.
Black flies (Diptera: Simuliidae) are haematophagous insects that can cause allergic reactions and act as vectors of pathogens. Although their saliva has been thought to contain a diverse array of physiologically active molecules, little information is available on antimicrobial factors in black fly salivary glands, especially no defensins have been reported so far.
A novel cationic defensin designated SibaDef was purified using reverse phase high-performance liquid chromatography (RP-HPLC) from the salivary glands of the black fly Simulium bannaense. The amino acid sequence of SibaDef was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-positive bacteria Staphylococcus aureus or Bacillus subtilis treated with SibaDef were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the expression of SibaDef mRNA in whole bodies of insects after oral infection with the bacteria S. aureus or B. subtilis.
Surprisingly, the phylogenetic analysis of defensin-related amino acid sequences demonstrated that SibaDef is most closely related to defensins from the human body louse Pediculus humanus corporis (Anoplura: Pediculidae), rather than to other dipteran defensins. SibaDef showed potent antimicrobial activities against Gram-positive bacteria with minimal inhibitory concentrations (MICs) ranging from 0.83 μM to 2.29 μM. SEM analysis indicated that SibaDef killed microorganisms through the disruption of cell membrane integrity. The transcript levels of SibaDef in the bacteria-immunized flies increased with the time course, reaching maximum at 36 h and then slowly decreased from that time point.
Our results indicate that SibaDef is involved in the innate humoral response of the black fly S. bannaense, and it might play a significant role in the defence against microorganisms in both sugar and blood meals.
PMCID: PMC4324660  PMID: 25649358
Insect; Antimicrobial peptide; Defensin; Salivary gland; Black fly; Simulium bannaense
19.  The Relationship between Water Temperature and the Development Cycle Beginning and Duration in Three Black Fly Species 
Understanding environmental factors affecting the timing and rate of animal development, as well as the factors that cause their effects, is of great importance. The purpose of this study was to establish the relationship between the onset and duration of the development from egg to pupal stage and water temperature in three black fly (Diptera: Simuliidae) species: Simulium (Simulium) reptans (Linnaeus 1758), Simulium (Byssodon) maculatum (Meigen 1804), Simulium (Boophthora) erythrocephalum (De Geer 1776). The study was based on surveys conducted between April and June of 1998–2010. The water temperature on the day of larval eclosion had no statistically significant impact on the beginning of development in any of the three species studied. The date when water temperature in the river reaches a certain value is important to the initiation of development in some black fly species. The present study revealed that the most important dates to the beginning of development of S. reptans black flies are when water temperature rises above 5° C, 7° C, and 10° C, while pivotal dates to the development of S. maculatum are when water temperature exceeds 4° C and 10° C. Water temperature most often exceeds the value important to the start of the development of these black fly species during March and April. The findings of the present study show that the hatching time of the two black fly species is also related to the mean water temperature in March and April. There were no statistically significant relations established between certain temperature dates and the beginning of larval development in S. erythrocephalum. Significant relations (p < 0.01) were found to exist between the duration of the development cycle from the first instar larva to pupa and the mean water temperature during the development period in S. reptans (r = -0.84; y = 53.088e-0.0806x, R2 = 0.70), S. maculatum (r = -0.82; y = 186.48e-0.1123x, R2 = 0.69) and S. erythrocephalum (r = -0.83; y = 58.768e-0.0652x, R2 = 0.70). The present study showed that the duration of development from the first instar larva to pupa in all the three black fly species studied was shorter when water temperatures during the development period were higher and longer when water temperatures were lower. The devised model of dependence between the duration of the studied black fly species' development and water temperature was verified experimentally.
PMCID: PMC3735164  PMID: 23879856
hatching; larvae; pupae; Simuliidae; temperature
20.  Description of a Nanobody-based Competitive Immunoassay to Detect Tsetse Fly Exposure 
PLoS Neglected Tropical Diseases  2015;9(2):e0003456.
Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts.
Methodology/Principal Findings
A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%).
We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.
Author Summary
Our previous studies have revealed that the saliva of the savannah tsetse fly (Glossina morsitans morsitans) and the main constituting Tsal proteins are sensitive immunological probes to detect contact with tsetse flies. A nanobody (Nb) library was generated against tsetse salivary gland proteins and used to select Nbs against the highly immunogenic Tsal proteins by a procedure of phage display and selection for binding onto the recombinant Tsal proteins. One Nb family was identified with the appropriate characteristics for the development of a competitive assay to detect Tsal-specific antibodies raised by the mammalian host when exposed to tsetse fly bites. In this immunoassay, exposure was detected by the inhibition of Nb binding by tsetse fly saliva induced antibodies in plasma. Evaluation of the competitive ELISA test using a set of porcine plasmas revealed an improved accuracy as compared to previously described tests. Moreover, the advantage of this assay is that it does not require adaptation to the sampled host species. We propose the Nb-based competitive ELISA as an additional tool to the indirect ELISA to serologically detect tsetse bite exposure and to monitor the impact of vector control programs and to detect re-invasion of cleared areas by tsetse flies on the African continent. In addition, the concept of using Nbs for the development of competitive antibody detection tests is innovative and broadens the scope of medical diagnostic applications of Nbs.
PMCID: PMC4320081  PMID: 25658871
21.  An insight into the salivary transcriptome and proteome of the soft tick and vector of epizootic bovine abortion, Ornithodoros coriaceus 
Journal of proteomics  2008;71(5):493-512.
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.
PMCID: PMC2617759  PMID: 18725333
Ornithodoros coriaceus; Ixodidae; Argasidae; Sialotranscriptome; Salivary gland transcriptome; Sialome; Tick salivary gland; Ixolaris
22.  Interruption of Onchocerca volvulus transmission in Northern Venezuela 
Parasites & Vectors  2013;6:289.
Onchocerciasis is caused by Onchocerca volvulus and transmitted by Simulium species (black flies). In the Americas, the infection has been previously described in 13 discrete regional foci distributed among six countries (Brazil, Colombia, Ecuador, Guatemala, Mexico and Venezuela) where more than 370,000 people are currently considered at risk. Since 2001, disease control in Venezuela has relied on the mass drug administration to the at-risk communities. This report provides empirical evidence of interruption of Onchocerca volvulus transmission by Simulium metallicum in 510 endemic communities from two Northern foci of Venezuela, after 10–12 years of 6-monthly Mectizan® (ivermectin) treatment to all the eligible residents.
In-depth entomologic and epidemiologic surveys were serially conducted from 2001–2012 in selected (sentinel and extra-sentinel) communities from the North-central (NC) and North-east (NE) onchocerciasis foci of Venezuela in order to monitor the impact of ivermectin treatment.
From 2007–2009, entomological indicators in both foci confirmed that 0 out of 112,637 S. metallicum females examined by PCR contained L3 infection in insect heads. The upper bound of the 95% confidence intervals of the infective rate of the vector reached values below 1% by 2009 (NC) and 2012 (NE). Additionally, after 14 (NC) and 22 (NE) rounds of treatment, the seasonal transmission potential (±UL CIs) of S. metallicum was under the critical threshold of 20 L3 per person per season. Serological analysis in school children < 15 years-old demonstrated that 0 out of 6,590 individuals were harboring antibodies to Ov-16. Finally, epidemiological surveys made during 2010 (NC) and 2012 (NE) showed no evidence of microfilariae in the skin and eyes of the population.
These results meet the WHO criteria for absence of parasite transmission and disease morbidity in these endemic areas which represent 91% of the population previously at-risk in the country. Consequently, the two Northern foci are currently under post-treatment onchocerciasis surveillance status in Venezuela.
PMCID: PMC3856516  PMID: 24499653
Onchocerciasis; Simulium metallicum; Interruption; Ivermectin; Venezuela
23.  Successful Interruption of Transmission of Onchocerca volvulus in the Escuintla-Guatemala Focus, Guatemala 
Elimination of onchocerciasis (river blindness) through mass administration of ivermectin in the six countries in Latin America where it is endemic is considered feasible due to the relatively small size and geographic isolation of endemic foci. We evaluated whether transmission of onchocerciasis has been interrupted in the endemic focus of Escuintla-Guatemala in Guatemala, based on World Health Organization criteria for the certification of elimination of onchocerciasis.
Methodology/Principal Findings
We conducted evaluations of ocular morbidity and past exposure to Onchocerca volvulus in the human population, while potential vectors (Simulium ochraceum) were captured and tested for O. volvulus DNA; all of the evaluations were carried out in potentially endemic communities (PEC; those with a history of actual or suspected transmission or those currently under semiannual mass treatment with ivermectin) within the focus. The prevalence of microfilariae in the anterior segment of the eye in 329 individuals (≥7 years old, resident in the PEC for at least 5 years) was 0% (one-sided 95% confidence interval [CI] 0–0.9%). The prevalence of antibodies to a recombinant O. volvulus antigen (Ov-16) in 6,432 school children (aged 6 to 12 years old) was 0% (one-sided 95% IC 0–0.05%). Out of a total of 14,099 S. ochraceum tested for O. volvulus DNA, none was positive (95% CI 0–0.01%). The seasonal transmission potential was, therefore, 0 infective stage larvae per person per season.
Based on these evaluations, transmission of onchocerciasis in the Escuintla-Guatemala focus has been successfully interrupted. Although this is the second onchocerciasis focus in Latin America to have demonstrated interruption of transmission, it is the first focus with a well-documented history of intense transmission to have eliminated O. volvulus.
Author Summary
Brought to the Americas from Africa by the slave trade, onchocerciasis is present in six countries in Latin America. The disease is caused by a round worm and is transmitted to humans by the bite of an infected black fly. Once in a human, the adult worms produce larvae that circulate through the body, causing itching or even blindness. Ivermectin, a drug that kills the larvae, is delivered by public health authorities in countries where the disease is present. If the larvae are killed, then the disease cannot be transmitted to more people. People living in the Escuintla-Guatemala focus, a region in Guatemala where the disease was common, have been taking ivermectin for many years. The Ministry of Health of Guatemala believes that onchocerciasis is no longer being transmitted in the area. To prove that there is no more transmission of the disease, the authors examined the eyes of residents of the area to see if they could find any evidence of the worms. They also conducted analyses of blood in school children to see if they had ever been exposed to the worm, and they caught thousands of black flies and tested them to see if they were infected. These evaluations found no evidence of transmission of the disease in the Escuintla-Guatemala focus. As a result, local public health authorities can stop giving ivermectin and invest their human resources in other important diseases.
PMCID: PMC2656640  PMID: 19333366
24.  Hyperreactive Onchocerciasis is Characterized by a Combination of Th17-Th2 Immune Responses and Reduced Regulatory T Cells 
Clinical manifestations in onchocerciasis range from generalized onchocerciasis (GEO) to the rare but severe hyperreactive (HO)/sowda form. Since disease pathogenesis is associated with host inflammatory reactions, we investigated whether Th17 responses could be related to aggravated pathology in HO. Using flow cytometry, filarial-specific cytokine responses and PCR arrays, we compared the immune cell profiles, including Th subsets, in individuals presenting the two polar forms of infection and endemic normals (EN). In addition to elevated frequencies of memory CD4+ T cells, individuals with HO showed accentuated Th17 and Th2 profiles but decreased CD4+CD25hiFoxp3+ regulatory T cells. These profiles included increased IL-17A+, IL-4+, RORC2+ and GATA3+CD4+ T cell populations. Flow cytometry data was further confirmed using a PCR array since Th17-related genes (IL-17 family members, IL-6, IL-1β and IL-22) and Th2-related (IL-4, IL-13, STAT6) genes were all significantly up-regulated in HO individuals. In addition, stronger Onchocerca volvulus-specific Th2 responses, especially IL-13, were observed in vitro in hyperreactive individuals when compared to GEO or EN groups. This study provides initial evidence that elevated frequencies of Th17 and Th2 cells form part of the immune network instigating the development of severe onchocerciasis.
Author Summary
Onchocerciasis, also known as river blindness is a tropical disease causing health and socioeconomic problems in endemic communities especially sub-Saharan Africa. The disease is transmitted by a filarial nematode called Onchocerca volvulus, which is spread by the bite of infected Simulium black flies. Characteristic disease symptoms include dermatological disorders and eye lesions that can lead to blindness. Two polar forms of clinical manifestations can occur: generalized onchocerciasis (GEO) presenting mild skin disease or the hyperreactive form (HO) exhibiting severe skin disorders and inflammation. The immunological determinants behind such disease polarization are still not fully clarified. Here, we compared the immune profiles of individuals presenting these two polar forms with those of endemic normals (EN): individuals who have no clinical or parasitological evidence of infection despite ongoing exposure to the infectious agent. We could show that HO individuals, in contrast to GEO and EN, simultaneously presented elevated Th17 and Th2 profiles which were accompanied by reduced numbers of Foxp3+ regulatory T cells. This study provides initial evidence that elevated frequencies of Th17 and Th2 cells form part of the immune network associated with severe onchocerciasis.
PMCID: PMC4288720  PMID: 25569210
25.  An Updated Insight into the Sialotranscriptome of Triatoma infestans: Developmental Stage and Geographic Variations 
Triatoma infestans is the main vector of Chagas disease in South America. As in all hematophagous arthropods, its saliva contains a complex cocktail that assists blood feeding by preventing platelet aggregation and blood clotting and promoting vasodilation. These salivary components can be immunologically recognized by their vector's hosts and targeted with antibodies that might disrupt blood feeding. These antibodies can be used to detect vector exposure using immunoassays. Antibodies may also contribute to the fast evolution of the salivary cocktail.
Salivary gland cDNA libraries from nymphal and adult T. infestans of breeding colonies originating from different locations (Argentina, Chile, Peru and Bolivia), and cDNA libraries originating from F1 populations of Bolivia, were sequenced using Illumina technology. Coding sequences (CDS) were extracted from the assembled reads, the numbers of reads mapped to these CDS, sequences were functionally annotated and polymorphisms determined.
Main findings/Significance
Over five thousand CDS, mostly full length or near full length, were publicly deposited on GenBank. Transcripts that were over 10-fold overexpressed from different geographical regions, or from different developmental stages were identified. Polymorphisms were mapped to derived coding sequences, and found to vary between developmental instars and geographic origin of the biological material. This expanded sialome database from T. infestans should be of assistance in future proteomic work attempting to identify salivary proteins that might be used as epidemiological markers of vector exposure, or proteins of pharmacological interest.
Author Summary
Triatoma infestans is the main vector of Chagas disease in South America. As in all hematophagous arthropods, its saliva contains a complex cocktail that assists blood feeding by preventing platelet aggregation and blood clotting and promoting vasodilation. These salivary components can be immunologically recognized by their hosts and targeted with antibodies that might disrupt blood feeding. The respective antibodies can be used to detect vector exposure using immunoassays. On the other hand, antibodies may also contribute to the fast evolution of the salivary cocktail. In this work, we attempted to identify variations in the salivary proteins of T. infestans using Illumina technology that allowed identification of over five thousand proteins based on over 300 million sequences obtained from ten salivary gland libraries. This expanded sialome database from T. infestans should be of assistance in future work attempting to identify salivary proteins that might be used as epidemiological markers of vector exposure, or proteins of pharmacological interest.
PMCID: PMC4256203  PMID: 25474469

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