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1.  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 http://exon.niaid.nih.gov/transcriptome/S_vittatum/T1/SV-tb1.zip and http://exon.niaid.nih.gov/transcriptome/S_vittatum/T2/SV-tb2.zip.
doi:10.1021/pr8008429
PMCID: PMC2778207  PMID: 19166301
Simulium vittatum; black fly; sialotranscriptomes; salivary gland transcriptome; sialome; proteome; hematophagy; onchocerciasis
2.  Identification of a Tsetse Fly Salivary Protein with Dual Inhibitory Action on Human Platelet Aggregation 
PLoS ONE  2010;5(3):e9671.
Background
Tsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated.
Methodology/Principal Findings
Here we report on the functional characterisation of a 5′nucleotidase-related (5′Nuc) saliva protein of the tsetse fly Glossina morsitans morsitans. This protein is encoded by a 1668 bp cDNA corresponding at the genomic level with a single-copy 4 kb gene that is exclusively transcribed in the tsetse salivary gland tissue. The encoded 5′Nuc protein is a soluble 65 kDa glycosylated compound of tsetse saliva with a dual anti-hemostatic action that relies on its combined apyrase activity and fibrinogen receptor (GPIIb/IIIa) antagonistic properties. Experimental evidence is based on the biochemical and functional characterization of recombinant protein and on the successful silencing of the 5′nuc translation in the salivary gland by RNA interference (RNAi). Refolding of a 5′Nuc/SUMO-fusion protein yielded an active apyrase enzyme with Km and Vmax values of 43±4 µM and 684±49 nmol Pi/min×mg for ATPase and 49±11 µM and 177±37 nmol Pi/min×mg for the ADPase activity. In addition, recombinant 5′Nuc was found to bind to GPIIb/IIIa with an apparent KD of 92±25 nM. Consistent with these features, 5′Nuc potently inhibited ADP-induced thrombocyte aggregation and even caused disaggregation of ADP-triggered human platelets. The importance of 5′Nuc for the tsetse fly hematophagy was further illustrated by specific RNAi that reduced the anti-thrombotic activities in saliva by approximately 50% resulting in a disturbed blood feeding process.
Conclusions/Significance
These data show that this 5′nucleotidase-related apyrase exhibits GPIIb/IIIa antagonistic properties and represents a key thromboregulatory compound of tsetse fly saliva.
doi:10.1371/journal.pone.0009671
PMCID: PMC2843633  PMID: 20351782
3.  An Insight into the Sialotranscriptome of the Cat Flea, Ctenocephalides felis 
PLoS ONE  2012;7(9):e44612.
Background
Saliva of hematophagous arthropods contains a diverse mixture of compounds that counteracts host hemostasis. Immunomodulatory and antiinflammatory components are also found in these organisms' saliva. Blood feeding evolved at least ten times within arthropods, providing a scenario of convergent evolution for the solution of the salivary potion. Perhaps because of immune pressure from hosts, the salivary proteins of related organisms have considerable divergence, and new protein families are often found within different genera of the same family or even among subgenera. Fleas radiated with their vertebrate hosts, including within the mammal expansion initiated 65 million years ago. Currently, only one flea species–the rat flea Xenopsylla cheopis–has been investigated by means of salivary transcriptome analysis to reveal salivary constituents, or sialome. We present the analysis of the sialome of cat flea Ctenocephaides felis.
Methodology and Critical Findings
A salivary gland cDNA library from adult fleas was randomly sequenced, assembled, and annotated. Sialomes of cat and rat fleas have in common the enzyme families of phosphatases (inactive), CD-39-type apyrase, adenosine deaminases, and esterases. Antigen-5 members are also common to both sialomes, as are defensins. FS-I/Cys7 and the 8-Cys families of peptides are also shared by both fleas and are unique to these organisms. The Gly-His-rich peptide similar to holotricin was found only in the cat flea, as were the abundantly expressed Cys-less peptide and a novel short peptide family.
Conclusions/Significance
Fleas, in contrast to bloodsucking Nematocera (mosquitoes, sand flies, and black flies), appear to concentrate a good portion of their sialome in small polypeptides, none of which have a known function but could act as inhibitors of hemostasis or inflammation. They are also unique in expansion of a phosphatase family that appears to be deficient of enzyme activity and has an unknown function.
doi:10.1371/journal.pone.0044612
PMCID: PMC3458046  PMID: 23049752
4.  The Influence of Sex and Fly Species on the Development of Trypanosomes in Tsetse Flies 
Unlike other dipteran disease vectors, tsetse flies of both sexes feed on blood and transmit pathogenic African trypanosomes. During transmission, Trypanosoma brucei undergoes a complex cycle of proliferation and development inside the tsetse vector, culminating in production of infective forms in the saliva. The insect manifests robust immune defences throughout the alimentary tract, which eliminate many trypanosome infections. Previous work has shown that fly sex influences susceptibility to trypanosome infection as males show higher rates of salivary gland (SG) infection with T. brucei than females. To investigate sex-linked differences in the progression of infection, we compared midgut (MG), proventriculus, foregut and SG infections in male and female Glossina morsitans morsitans. Initially, infections developed in the same way in both sexes: no difference was observed in numbers of MG or proventriculus infections, or in the number and type of developmental forms produced. Female flies tended to produce foregut migratory forms later than males, but this had no detectable impact on the number of SG infections. The sex difference was not apparent until the final stage of SG invasion and colonisation, showing that the SG environment differs between male and female flies. Comparison of G. m. morsitans with G. pallidipes showed a similar, though less pronounced, sex difference in susceptibility, but additionally revealed very different levels of trypanosome resistance in the MG and SG. While G. pallidipes was more refractory to MG infection, a very high proportion of MG infections led to SG infection in both sexes. It appears that the two fly species use different strategies to block trypanosome infection: G. pallidipes heavily defends against initial establishment in the MG, while G. m. morsitans has additional measures to prevent trypanosomes colonising the SG, particularly in female flies. We conclude that the tsetse-trypanosome interface works differently in G. m. morsitans and G. pallidipes.
Author Summary
In tropical Africa human and livestock diseases caused by parasitic trypanosomes are transmitted by bloodsucking tsetse flies. In the fly, trypanosomes undergo a complex cycle of proliferation and development during their remarkable journey from the midgut to the salivary glands. At every step of the way, the flies mount robust immune defences against trypanosome infection and consequently most flies fail to develop a transmissible infection. Previous work has shown a sex difference in the numbers of salivary gland infections with Trypanosoma brucei: male flies are more susceptible to salivary gland infection than females. Here we explored possible reasons for this. Infections developed in the same way in both male and female flies until the final stage of salivary gland invasion and colonisation. We conclude that the salivary gland environment in the female fly is much more inhospitable for trypanosomes, perhaps because of a greater immune response. Comparison of two different tsetse species showed very different levels of trypanosome resistance in the midgut and salivary glands.
doi:10.1371/journal.pntd.0001515
PMCID: PMC3279344  PMID: 22348165
5.  A further insight into the sialome of the tropical bont tick, Amblyomma variegatum 
BMC Genomics  2011;12:136.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2164-12-136
PMCID: PMC3060141  PMID: 21362191
6.  Expression of chemosensory proteins in the tsetse fly Glossina morsitans morsitans is related to female host-seeking behaviour 
Insect Molecular Biology  2011;21(1):41-48.
Chemosensory proteins (CSPs) are a class of soluble proteins present in high concentrations in the sensilla of insect antennae. It has been proposed that they play an important role in insect olfaction by mediating interactions between odorants and odorant receptors. Here we report, for the first time, the presence of five CSP genes in the tsetse fly Glossina morsitans morsitans, a major vector transmitting nagana in livestock. Real-time quantitative reverse transcription PCR showed that three of the CSPs are expressed in antennae. One of them, GmmCSP2, is transcribed at a very high level and could be involved in olfaction. We also determined expression in the antennae of both males and females at different life stages and with different blood feeding regimes. The transcription of GmmCSP2 was lower in male antennae than in females, with a sharp increase in 10-week-old flies, 48 h after a bloodmeal. Thus there is a clear relationship between CSP gene transcription and host searching behaviour. Genome annotation and phylogenetic analyses comparing G. morsitans morsitans CSPs with those of other Diptera showed rapid evolution after speciation of mosquitoes.
doi:10.1111/j.1365-2583.2011.01114.x
PMCID: PMC3664020  PMID: 22074189
chemosensory protein; tsetse fly; gene expression; trypanosomiasis; nagana
7.  An insight into the sialome of blood feeding Nematocera 
Within the Diptera and outside the suborder Brachycera, the blood feeding habit occurred at least twice, producing the present day sand flies, and the Culicomorpha, including the mosquitoes (Culicidae), black flies (Simulidae), biting midges (Ceratopogonidae) and frog feeding flies (Corethrellidae). Alternatives to this scenario are also discussed. Successful blood feeding requires adaptations to antagonize the vertebrate's mechanisms of blood clotting, platelet aggregation, vasoconstriction, pain and itching, which are triggered by tissue destruction and immune reactions to insect products. Saliva of these insects provides a complex pharmacological armamentarium to block these vertebrate reactions. With the advent of transcriptomics, the sialomes (from the Greek word sialo=saliva) of at least two species of each of these families have been studied (except for the frog feeders), allowing an insight into the diverse pathways leading to today's salivary composition within the Culicomorpha, having the sand flies as an outgroup. This review catalogs 1,288 salivary proteins in 10 generic classes comprising over 150 different protein families, most of which we have no functional knowledge. These proteins and many sequence comparisons are displayed in a hyperlinked spreadsheet that hopefully will stimulate and facilitate the task of functional characterization of these proteins, and their possible use as novel pharmacological agents and epidemiological markers of insect vector exposure.
doi:10.1016/j.ibmb.2010.08.002
PMCID: PMC2950210  PMID: 20728537
8.  Where, When and Why Do Tsetse Contact Humans? Answers from Studies in a National Park of Zimbabwe 
Background
Sleeping sickness, also called human African trypanosomiasis, is transmitted by the tsetse, a blood-sucking fly confined to sub-Saharan Africa. The form of the disease in West and Central Africa is carried mainly by species of tsetse that inhabit riverine woodland and feed avidly on humans. In contrast, the vectors for the East and Southern African form of the disease are usually savannah species that feed mostly on wild and domestic animals and bite humans infrequently, mainly because the odours produced by humans can be repellent. Hence, it takes a long time to catch many savannah tsetse from people, which in turn means that studies of the nature of contact between savannah tsetse and humans, and the ways of minimizing it, have been largely neglected.
Methodology/Principal Findings
The savannah tsetse, Glossina morsitans morsitans and G. pallidipes, were caught from men in the Mana Pools National park of Zimbabwe. Mostly the catch consisted of young G. m. morsitans, with little food reserve. Catches were increased by 4–8 times if the men were walking, not stationary, and increased about ten times more if they rode on a truck at 10 km/h. Catches were unaffected if the men used deodorant or were baited with artificial ox odour, but declined by about 95% if the men were with an ox. Surprisingly, men pursuing their normal daily activities were bitten about as much when in or near buildings as when in woodland. Catches from oxen and a standard ox-like trap were poor indices of the number and physiological state of tsetse attacking men.
Conclusion/Significance
The search for new strategies to minimize the contact between humans and savannah tsetse should focus on that occurring in buildings and vehicles. There is a need to design a man-like trap to help to provide an index of sleeping sickness risk.
Author Summary
To identify where to look for new strategies to limit the contact between tsetse and humans, and so reduce the transmission of sleeping sickness, we examined various factors influencing the numbers of tsetse caught from men. Catches were increased if the men walked, or travelled a little faster on a truck, and were reduced if an ox were nearby. There was no effect of making the men smell like an ox, or of spraying the men with deodorant. Intriguingly, while it is usually assumed tacitly that most human/tsetse contact occurs when people enter the normal woodland habitat of the flies, we found that just as much contact occurred due to tsetse visiting human habitations. We also operated standard ox-like traps to assess whether their catches could be used as indices of the biting rate on humans, but found too many distinctions between catches from these traps and people. Overall, it appeared that the search for means of restricting human/fly contact should focus on that occurring in buildings and vehicles, and that attempts should be made to design traps that are more man-like.
doi:10.1371/journal.pntd.0001791
PMCID: PMC3429383  PMID: 22953013
9.  Lipophorin acts as a shuttle of lipids to the milk gland during tsetse fly pregnancy 
Journal of insect physiology  2011;57(11):1553-1561.
During pregnancy in the viviparous tsetse fly, lipid mobilization is essential for the production of milk to feed the developing intrauterine larva. Lipophorin (Lp) functions as the major lipid transport protein in insects and closely-related arthropods. In this study, we assessed the role of Lp and the lipophorin receptor (LpR) in the lipid mobilization process during tsetse reproduction. We identified single gene sequences for GmmLp and GmmLpR from the genome of Glossina morsitans morsitans, and measured spatial and temporal expression of gmmlp and gmmlpr during the female reproductive cycle. Our results show that expression of gmmlp is specific to the adult fat body and larvae. In the adult female, gmmlp expression is constitutive. However transcript levels increase in the larva as it matures within the mother’s uterus, reaching peak expression just prior to parturition. GmmLp was detected in the hemolymph of pregnant females and larvae, but not in the uterine fluid or larval gut contents ruling out the possibility of direct transfer of GmmLp from mother to offspring. Transcripts for gmmlpr were detected in the head, ovaries, midgut, milk gland/fat body, ovaries and developing larva. Levels of gmmlpr remain stable throughout the first and second gonotrophic cycles with a slight dip observed during the first gonotrophic cycle. GmmLpR was detected in multiple tissues, including the midgut, fat body, milk gland, spermatheca and head. Knockdown of gmmlp by RNA interference resulted in reduced hemolymph lipid levels, delayed oocyte development and extended larval gestation. Similar suppresion of gmmlpr did not significantly reduce hemolymph lipid levels or oogenesis duration, but did extend the duration of larval development. Thus, GmmLp and GmmLpR function as the primary shuttle for lipids originating from the midgut and fat body to the ovaries and milk gland to supply resources for developing oocytes and larval nourishment, respectively. Once in the milk gland however, lipids are apparently transferred into the developing larva not by lipophorin but by another carrier lipoprotein.
doi:10.1016/j.jinsphys.2011.08.009
PMCID: PMC3209505  PMID: 21875592
Lipid movement; lipophorin; tsetse development; Glossina
10.  Interspecific Transfer of Bacterial Endosymbionts between Tsetse Fly Species: Infection Establishment and Effect on Host Fitness▿  
Applied and Environmental Microbiology  2006;72(11):7013-7021.
Tsetse flies (Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera (Glossina fuscipes fuscipes and Glossina morsitans morsitans). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.
doi:10.1128/AEM.01507-06
PMCID: PMC1636136  PMID: 16950907
11.  Post Eclosion Age Predicts the Prevalence of Midgut Trypanosome Infections in Glossina 
PLoS ONE  2011;6(11):e26984.
The teneral phenomenon, as observed in Glossina sp., refers to the increased susceptibility of the fly to trypanosome infection when the first bloodmeal taken is trypanosome-infected. In recent years, the term teneral has gradually become synonymous with unfed, and thus fails to consider the age of the newly emerged fly at the time the first bloodmeal is taken. Furthermore, conflicting evidence exists of the effect of the age of the teneral fly post eclosion when it is given the infected first bloodmeal in determining the infection prevalence. This study demonstrates that it is not the feeding history of the fly but rather the age (hours after eclosion of the fly from the puparium) of the fly when it takes the first (infective) bloodmeal that determines the level of fly susceptibility to trypanosome infection. We examine this phenomenon in male and female flies from two distinct tsetse clades (Glossina morsitans morsitans and Glossina palpalis palpalis) infected with two salivarian trypanosome species, Trypanosoma (Trypanozoon) brucei brucei and Trypanosoma (Nannomonas) congolense using Fisher's exact test to examine differences in infection rates. Teneral tsetse aged less than 24 hours post-eclosion (h.p.e.) are twice as susceptible to trypanosome infection as flies aged 48 h.p.e. This trend is conserved across sex, vector clade and parasite species. The life cycle stage of the parasite fed to the fly (mammalian versus insect form trypanosomes) does not alter this age-related bias in infection. Reducing the numbers of parasites fed to 48 h.p.e., but not to 24 h.p.e. flies, increases teneral refractoriness. The importance of this phenomenon in disease biology in the field as well as the necessity of employing flies of consistent age in laboratory-based infection studies is discussed.
doi:10.1371/journal.pone.0026984
PMCID: PMC3210762  PMID: 22087240
12.  An insight into the transcriptome and proteome of the salivary gland of the stable fly, Stomoxys calcitrans 
Adult stable flies are blood feeders, a nuisance, and mechanical vectors of veterinary diseases. To enable efficient feeding, blood sucking insects have evolved a sophisticated array of salivary compounds to disarm their host's hemostasis and inflammatory reaction. While the sialomes of several blood sucking Nematocera flies have been described, no thorough description has been made so far of any Brachycera, except for a detailed proteome analysis of a tabanid (Xu et al., 2008). In this work we provide an insight into the sialome of the muscid Stomoxys calcitrans, revealing a complex mixture of serine proteases, endonucleases, Kazal-containing peptides, anti-thrombins, antigen-5 related proteins, antimicrobial peptides, and the usual finding of mysterious secreted peptides that have no known partners, and may reflect the very fast evolution of salivary proteins due to the vertebrate host immune pressure. Supplemental tables S1 and S2 can be downloaded from http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T1/Sc-tb1-web.xls and http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T2/Sc-tb2-web.xls.
doi:10.1016/j.ibmb.2009.06.004
PMCID: PMC2737351  PMID: 19576987
Salivary glands; stable fly; hematophagy; sialome; cDNA library; proteome
13.  A Deep Insight into the Sialotranscriptome of the Gulf Coast Tick, Amblyomma maculatum 
PLoS ONE  2011;6(12):e28525.
Background
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.
Methodology/Principal Findings
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.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0028525
PMCID: PMC3244413  PMID: 22216098
14.  An insight into the sialotranscriptome and proteome of the coarse bontlegged tick, Hyalomma marginatum rufipes 
Journal of proteomics  2011;74(12):2892-2908.
Ticks are mites specialized in acquiring blood from vertebrates as their sole source of food and are important disease vectors to humans and animals. Among the specializations required for this peculiar diet, ticks evolved a sophisticated salivary potion that can disarm their host’s hemostasis, inflammation, and immune reactions. Previous transcriptome analysis of tick salivary proteins has revealed many new protein families indicative of fast evolution, possibly due to host immune pressure. The hard ticks (family Ixodidae) are further divided into two basal groups, of which the Metastriata have 11 genera. While salivary transcriptomes and proteomes have been described for some of these genera, no tick of the genus Hyalomma has been studied so far. The analysis of 2,084 expressed sequence tags (EST) from a salivary gland cDNA library allowed an exploration of the proteome of this tick species by matching peptide ions derived from MS/MS experiments to this data set. We additionally compared these MS/MS derived peptide sequences against the proteins from the bovine host, finding many host proteins in the salivary glands of this tick. This annotated data set can assist the discovery of new targets for anti-tick vaccines as well as help to identify pharmacologically active proteins.
doi:10.1016/j.jprot.2011.07.015
PMCID: PMC3215792  PMID: 21851864
Tick; hematophagy; salivary glands; sialome
15.  The Salivary Secretome of the Tsetse Fly Glossina pallidipes (Diptera: Glossinidae) Infected by Salivary Gland Hypertrophy Virus 
Background
The competence of the tsetse fly Glossina pallidipes (Diptera; Glossinidae) to acquire salivary gland hypertrophy virus (SGHV), to support virus replication and successfully transmit the virus depends on complex interactions between Glossina and SGHV macromolecules. Critical requisites to SGHV transmission are its replication and secretion of mature virions into the fly's salivary gland (SG) lumen. However, secretion of host proteins is of equal importance for successful transmission and requires cataloging of G. pallidipes secretome proteins from hypertrophied and non-hypertrophied SGs.
Methodology/Principal Findings
After electrophoretic profiling and in-gel trypsin digestion, saliva proteins were analyzed by nano-LC-MS/MS. MaxQuant/Andromeda search of the MS data against the non-redundant (nr) GenBank database and a G. morsitans morsitans SG EST database, yielded a total of 521 hits, 31 of which were SGHV-encoded. On a false discovery rate limit of 1% and detection threshold of least 2 unique peptides per protein, the analysis resulted in 292 Glossina and 25 SGHV MS-supported proteins. When annotated by the Blast2GO suite, at least one gene ontology (GO) term could be assigned to 89.9% (285/317) of the detected proteins. Five (∼1.8%) Glossina and three (∼12%) SGHV proteins remained without a predicted function after blast searches against the nr database. Sixty-five of the 292 detected Glossina proteins contained an N-terminal signal/secretion peptide sequence. Eight of the SGHV proteins were predicted to be non-structural (NS), and fourteen are known structural (VP) proteins.
Conclusions/Significance
SGHV alters the protein expression pattern in Glossina. The G. pallidipes SG secretome encompasses a spectrum of proteins that may be required during the SGHV infection cycle. These detected proteins have putative interactions with at least 21 of the 25 SGHV-encoded proteins. Our findings opens venues for developing novel SGHV mitigation strategies to block SGHV infections in tsetse production facilities such as using SGHV-specific antibodies and phage display-selected gut epithelia-binding peptides.
Author Summary
Tsetse fly (Diptera; Glossinidae) transmits two devastating diseases to farmers (human African Trypanosomiasis; HAT) and their livestock (Animal African Trypanosomiasis; AAT) in 37 sub-Saharan African countries. During the rainy seasons, vast areas of fertile, arable land remain uncultivated as farmers flee their homes due to the presence of tsetse. Available drugs against trypanosomiasis are ineffective and difficult to administer. Control of the tsetse vector by Sterile Insect Technique (SIT) has been effective. This method involves repeated release of sterilized males into wild tsetse populations, which compete with wild type males for females. Upon mating, there is no offspring, leading to reduction in tsetse populations and thus relief from trypanosomiasis. The SIT method requires large-scale tsetse rearing to produce sterile males. However, tsetse colony productivity is hampered by infections with the salivary gland hypertrophy virus, which is transmitted via saliva as flies take blood meals during membrane feeding and often leads to colony collapse. Here, we investigated the salivary gland secretome proteins of virus-infected tsetse to broaden our understanding of virus infection, transmission and pathology. By this approach, we obtain insight in tsetse-hytrosavirus interactions and identified potential candidate proteins as targets for developing biotechnological strategies to control viral infections in tsetse colonies.
doi:10.1371/journal.pntd.0001371
PMCID: PMC3222630  PMID: 22132244
16.  Molecular aspects of viviparous reproductive biology of the tsetse fly (Glossina morsitans morsitans): Regulation of yolk and milk gland protein synthesis 
Journal of insect physiology  2006;52(11-12):1128-1136.
Tsetse fly (Diptera: Glossinidae) viviparous reproductive physiology remains to be explored at the molecular level. Adult females carry their young in utero for the duration of embryonic and larval development, all the while supplying their offspring with nutrients in the form of a “milk” substance secreted from a modified accessory gland. Flies give birth to fully developed third instar larvae that pupariate shortly after birth. Here, we describe the spatial and temporal expression dynamics of two reproduction-associated genes and their products synthesized during the first and second gonotrophic cycles. The proteins studied include a putative yolk protein, Glossina morsitans morsitans yolk protein 1 (GmmYP1) and the major protein found in tsetse “milk” secretions (Glossina morsitans morsitans milk gland protein, GmmMGP). Developmental stage and tissue-specific expression of GmmYP1 show its presence exclusively in the reproductive tract of the fly during oogenesis, suggesting that GmmYP1 acts as a vitellogenic protein. Transcripts for GmmMGP are present only in the milk gland tissue and increase in coordination with the process of larvigenesis. Similarly, GmmMGP can be detected at the onset of larvigenesis in the milk gland, and is present during the full duration of pregnancy. Expression of GmmMGP is restricted to the adult stage and is not detected in the immature developmental stages. These phenomena indicate that the protein is transferred from mother to larvae as nourishment during its development. These results demonstrate that both GmmYP1 and GmmMGP are involved in tsetse reproductive biology, the former associated with the process of oogenesis and the latter with larvigenesis.
doi:10.1016/j.jinsphys.2006.07.007
PMCID: PMC1779500  PMID: 17046784
Viviparous reproduction; Oogenesis; Larvagenesis; Tsetse; Milk gland
17.  An insight into the sialome of the bed bug, Cimex lectularius 
Journal of proteome research  2010;9(8):3820-3831.
The evolution of insects to a blood diet leads to the development of a saliva that antagonizes their hosts' hemostasis and inflammation. Hemostasis and inflammation are redundant processes, and thus a complex salivary potion comprised of dozens or near one hundred different polypeptides is commonly found by transcriptome or proteome analysis of these organisms. Several insect orders or families evolved independently to hematophagy creating unique salivary potions in the form of novel pharmacological use of endogenous substances, and in the form of unique proteins not matching other known proteins, these probably arriving by fast evolution of salivary proteins as they evade their hosts' immune response. In this work we present a preliminary description of the sialome (from the Greek Sialo = saliva) of the common bed bug Cimex lectularius, the first such work from a member of the Cimicidae family. This manuscript is a guide for the supplemental database files http://exon.niaid.nih.gov/transcriptome/C_lectularius/S1/Cimex-S1.zip and http://exon.niaid.nih.gov/transcriptome/C_lectularius/S2/Cimex-S2.xls
doi:10.1021/pr1000169
PMCID: PMC2917537  PMID: 20441151
Bedbug; saliva; salivary transcriptome; salivary proteome
18.  Infections with Immunogenic Trypanosomes Reduce Tsetse Reproductive Fitness: Potential Impact of Different Parasite Strains on Vector Population Structure 
The parasite Trypanosoma brucei rhodesiense and its insect vector Glossina morsitans morsitans were used to evaluate the effect of parasite clearance (resistance) as well as the cost of midgut infections on tsetse host fitness. Tsetse flies are viviparous and have a low reproductive capacity, giving birth to only 6–8 progeny during their lifetime. Thus, small perturbations to their reproductive fitness can have a major impact on population densities. We measured the fecundity (number of larval progeny deposited) and mortality in parasite-resistant tsetse females and untreated controls and found no differences. There was, however, a typanosome-specific impact on midgut infections. Infections with an immunogenic parasite line that resulted in prolonged activation of the tsetse immune system delayed intrauterine larval development resulting in the production of fewer progeny over the fly's lifetime. In contrast, parasitism with a second line that failed to activate the immune system did not impose a fecundity cost. Coinfections favored the establishment of the immunogenic parasites in the midgut. We show that a decrease in the synthesis of Glossina Milk gland protein (GmmMgp), a major female accessory gland protein associated with larvagenesis, likely contributed to the reproductive lag observed in infected flies. Mathematical analysis of our empirical results indicated that infection with the immunogenic trypanosomes reduced tsetse fecundity by 30% relative to infections with the non-immunogenic strain. We estimate that a moderate infection prevalence of about 26% with immunogenic parasites has the potential to reduce tsetse populations. Potential repercussions for vector population growth, parasite–host coevolution, and disease prevalence are discussed.
Author Summary
In many cases, parasites adapt to their hosts' biology over time and the extent of their harmful effects gradually diminishes. Insect-transmitted parasites such as African trypanosomes, however, are unusually pathogenic for their mammalian hosts because they rely on their invertebrate hosts for transmission to the next mammalian host. To ensure their maximum transmission, it is essential that parasite infections do not compromise insect host's fitness traits, including longevity and host-finding ability. Our results in tsetse indicate that, as theory predicts, trypanosome infections do not reduce host longevity. Instead, they divert host resources from reproduction and can reduce reproductive output by as much as 30%. Such loss of reproductive fitness occurs as a result of the induction of tsetse's immune responses. A closely related non-immunogenic parasite line does not induce host responses and does not compromise host fecundity. It is possible that host immune responses are needed in the case of the immunogenic line to control the parasite density to prevent excessive host damage. Because tsetse are viviparous and each adult female typically gives rise to only few progeny during their lifetime, even modest costs on reproduction can have a significant impact on host abundance. Our model predicts that if the prevalence of immunogenic parasite infections in tsetse populations reaches over 26%, they begin to have a negative impact on population growth rate. Infection rates as high as 30% have been reported with trypanosomes in the field. Our laboratory findings coupled with our modeling studies now provide a framework to investigate the status of co-infections, host immune activation processes, fecundity outcomes, transmission dynamics, and host virulence phenotypes in natural tsetse–trypanosome populations.
doi:10.1371/journal.pntd.0000192
PMCID: PMC2265429  PMID: 18335067
19.  Tsetse-Wolbachia symbiosis: Comes of age and has great potential for pest and disease control 
Journal of invertebrate pathology  2012;112(0):S94-103.
Tsetse flies (Diptera: Glossinidae) are the sole vectors of African trypanosomes, the causative agent of sleeping sickness in human and nagana in animals. Like most eukaryotic organisms, Glossina species have established symbiotic associations with bacteria. Three main symbiotic bacteria have been found in tsetse flies: Wigglesworthia glossinidia, an obligate symbiotic bacterium, the secondary endosymbiont Sodalis glossinidius and the reproductive symbiont Wolbachia pipientis. In the present review, we discuss recent studies on the detection and characterization of Wolbachia infections in Glossina species, the horizontal transfer of Wolbachia genes to tsetse chromosomes, the ability of this symbiont to induce cytoplasmic incompatibility in Glossina morsitans morsitans and also how new environment-friendly tools for disease control could be developed by harnessing Wolbachia symbiosis.
doi:10.1016/j.jip.2012.05.010
PMCID: PMC3772542  PMID: 22835476
Glossina; Wolbachia; Insect symbiosis; Sodalis; Wigglesworthia; Paratransgenesis
20.  Experimental evaluation of xenodiagnosis to detect trypanosomes at low parasitaemia levels in infected hosts 
Parasite  2011;18(4):295-302.
In Human African Trypanosomosis (HAT) endemic areas, there are a number of subjects that are positive to serological tests but in whom trypanosomes are difficult to detect with the available parasitological tests. In most cases and particularly in West Africa, these subjects remain untreated, thus posing a fundamental problem both at the individual level (because of a possible lethal evolution of the disease) and at the epidemiological level (since they are potential reservoirs of trypanosomes). Xenodiagnosis may constitute an alternative for this type of cases. The objective of this study was to update the use of xenodiagnosis to detect trypanosomes in infected host characterized by low parasitaemia levels. This was carried out experimentally by infecting cattle and pigs with Trypanosoma congolense and T. brucei gambiense respectively, and by feeding tsetse flies (Glossina morsitans submorsitans and G. palpalis gambiensis, from the CIRDES colonies) on these animals at a time when the observed blood parasitaemia were low or undetectable by the classical microscopic parasitological tests used for the monitoring of infected animals. Our results showed that: i) the G. p. gambiensis colony at CIRDES could not be infected with the T. b. gambiense stocks used; ii) midgut infections of G. m. submorsitans were observed with both T. congolense and T. b. gambiense; iii) xenodiagnosis remains positive even at very low blood parasitaemia for both T. congolense and T. b. gambiense; and iv) to implement T. b. gambiense xenodiagnosis, batches of 20 G. m. submorsitans should be dissected two days after the infective meal. These results constitute a first step toward a possible implementation of xenodiagnosis to better characterize the parasitological status of seropositive individuals and the modalities of parasite transmission in HAT foci.
doi:10.1051/parasite/2011184295
PMCID: PMC3677595  PMID: 22091459
xenodiagnosis; experimental evaluation; HAT; tsetse; trypanosome; parasitaemia; xénodiagnostic; évaluation expérimentale; THA; glossine; trypanosome; parasitémie
21.  Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity 
PLoS ONE  2012;7(10):e47233.
Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.
doi:10.1371/journal.pone.0047233
PMCID: PMC3479092  PMID: 23110062
22.  Global Wolbachia prevalence, titer fluctuations and their potential of causing cytoplasmic incompatibilities in tsetse flies and hybrids of Glossina morsitans subgroup species 
Journal of Invertebrate Pathology  2013;112(Suppl 1):S104-S115.
Graphical abstract
Highlights
► VNTRs are highly diagnostic tools for fingerprinting Wolbachia in tsetse flies. ► Multiple infections, free and nuclear insertions into host chromosomes, do exist. ► Some infections can escape detection via hiding as low-titer infections. ► In hybrids Wolbachia can transform into pathogens by loss of replication control.
We demonstrate the high applicability of a novel VNTR-based (Variable-Number-Tandem-Repeat) molecular screening tool for fingerprinting Wolbachia-infections in tsetse flies. The VNTR-141 locus provides reliable and concise differentiation between Wolbachia strains deriving from Glossina morsitans morsitans, Glossina morsitans centralis, and Glossina brevipalpis. Moreover, we show that certain Wolbachia-infections in Glossina spp. are capable of escaping standard PCR screening methods by ‘hiding’ as low-titer infections below the detection threshold. By applying a highly sensitive PCR-blot technique to our Glossina specimen, we were able to enhance the symbiont detection limit substantially and, consequently, trace unequivocally Wolbachia-infections at high prevalence in laboratory-reared G. swynnertoni individuals. To our knowledge, Wolbachia-persistence was reported exclusively for field-collected samples, and at low prevalence only. Finally, we highlight the substantially higher Wolbachia titer levels found in hybrid Glossina compared to non-hybrid hosts and the possible impact of these titers on hybrid host fitness that potentially trigger incipient speciation in tsetse flies.
doi:10.1016/j.jip.2012.03.024
PMCID: PMC3625123  PMID: 22516306
Glossina; Wolbachia; Symbiont diversity; Inter-species hybrids; Speciation
23.  Tsetse Fly Saliva Accelerates the Onset of Trypanosoma brucei Infection in a Mouse Model Associated with a Reduced Host Inflammatory Response▿  
Infection and Immunity  2006;74(11):6324-6330.
Tsetse flies (Glossina sp.) are the vectors that transmit African trypanosomes, protozoan parasites that cause human sleeping sickness and veterinary infections in the African continent. These blood-feeding dipteran insects deposit saliva at the feeding site that enables the blood-feeding process. Here we demonstrate that tsetse fly saliva also accelerates the onset of a Trypanosoma brucei infection. This effect was associated with a reduced inflammatory reaction at the site of infection initiation (reflected by a decrease of interleukin-6 [IL-6] and IL-12 mRNA) as well as lower serum concentrations of the trypanocidal cytokine tumor necrosis factor. Variant-specific surface glycoprotein-specific antibody isotypes immunoglobulin M (IgM) and IgG2a, implicated in trypanosome clearance, were not suppressed. We propose that tsetse fly saliva accelerates the onset of trypanosome infection by inhibiting local and systemic inflammatory responses involved in parasite control.
doi:10.1128/IAI.01046-06
PMCID: PMC1695494  PMID: 16954393
24.  Transfer of inoculum of Metarhizium anisopliae between adult Glossina morsitans morsitans and effects of fungal infection on blood feeding and mating behaviors 
Journal of Pest Science  2012;86(2):285-292.
The transfer of conidia of Metarhizium anisopliae between tsetse flies Glossina morsitans and the effects of fungal inoculation on mating and blood meal feeding behaviors were investigated in the laboratory. Male or female flies were inoculated with fungal conidia (“donors“) and allowed to pair with fungus-free mate of opposite sex (“recipients”) at 1-day-interval up to three mates. Fungus-treated male or female “donor” flies as well as their mates “recipients” died from fungal infection. However, mortality in male “recipient” flies declined with successive mating, from 82.5 to 32.5 %. Fungus-treated males readily located female flies and mating was successful in most cases comparable to the controls. There were no significant differences (P > 0.05) in mean duration of mating, number of jerking movements between fungus-treated and fungus-free males for all the mating lines, except in the number of jerking movements when male flies mated with the 3rd line female flies. Fungus-treated and fungus-free female flies previously mated with treated and non-treated males showed refractoriness during subsequent pairings. The number of fertile female flies was higher (P < 0.05) in fungus-free than in fungus-treated treatments, thus producing more pupae. High concentration of fungus (3.0 × 106 conidia ml−1) significantly (P < 0.05) reduced blood meal intake of flies. This study has shown that fungal infection does not affect the mating behavior of tsetse flies and fly-to-fly contamination does occur during matings. These are important attributes if entomopathogenic fungi have to be used in auto-dissemination strategy and be integrated into sterile insect technique.
doi:10.1007/s10340-012-0473-7
PMCID: PMC3656219  PMID: 23687485
Glossina morsitans morsitans; Metarhizium anisopliae; Entomopathogenic fungus; Tsetse; Mating behavior; Infection; Blood meal; Transfer of inoculum; Reproduction
25.  Repellent Properties of δ-Octalactone Against the Tsetse Fly, Glossina morsitans Morsitans  
δ-octalactone, produced by several Bovidae, has been suggested as a potential repellant of tsetse fly attack. Racemic δ-octalactone was synthesized via an abbreviated route. The product was assayed against 3-day old starved teneral female tsetse flies, Glossina morsitans morsitans Wiedemann (Diptera: Glossinidae), in a choice wind tunnel and found to be a potent tsetse repellent at doses ≥0.05 mg in 200 µl of paraffin oil (0.05 >p >0.01).
doi:10.1673/031.008.4301
PMCID: PMC3127398  PMID: 20298116
allomone; bioassay; choice wind tunnel; olfaction; racemic δ-octalactone synthesis

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