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1.  Tsetse GmmSRPN10 Has Anti-complement Activity and Is Important for Successful Establishment of Trypanosome Infections in the Fly Midgut 
The complement cascade in mammalian blood can damage the alimentary tract of haematophagous arthropods. As such, these animals have evolved their own repertoire of complement-inactivating factors, which are inadvertently exploited by blood-borne pathogens to escape complement lysis. Unlike the bloodstream stages, the procyclic (insect) stage of Trypanosoma brucei is highly susceptible to complement killing, which is puzzling considering that a tsetse takes a bloodmeal every 2–4 days. In this study, we identified four tsetse (Glossina morsitans morsitans) serine protease inhibitors (serpins) from a midgut expressed sequence tag (EST) library (GmmSRPN3, GmmSRPN5, GmmSRPN9 and GmmSRPN10) and investigated their role in modulating the establishment of a T. brucei infection in the midgut. Although not having evolved in a common blood-feeding ancestor, all four serpins have an active site sharing remarkable homology with the human complement C1-inhibitor serpin, SerpinG1. RNAi knockdown of individual GmmSRPN9 and GmmSRPN10 genes resulted in a significant decreased rate of infection by procyclic form T. brucei. Furthermore, recombinant GmmSRPN10 was both able to inhibit the activity of human complement-cascade serine proteases, C1s and Factor D, and to protect the in vitro killing of procyclic trypanosomes when incubated with complement-activated human serum. Thus, the secretion of serpins, which may be part of a bloodmeal complement inactivation system in tsetse, is used by procyclic trypanosomes to evade an influx of fresh trypanolytic complement with each bloodmeal. This highlights another facet of the complicated relationship between T. brucei and its tsetse vector, where the parasite takes advantage of tsetse physiology to further its chances of propagation and transmission.
Author Summary
Blood feeding arthropods are exploited by blood borne parasites as vectors of transmission. Trypanosoma brucei, a salivarian trypanosome species, must survive, migrate and differentiate in the tsetse until they become mature, mammalian-infective forms within the fly salivary glands. This constitutes a significant challenge to trypanosomes as the major parasite form colonising the tsetse midgut is sensitive to lysis by blood complement, which is introduced into the tsetse gut whenever the fly feeds. In this study, we show that T. brucei may avoid being eliminated by bloodmeal complement by benefitting from a complement-inhibiting enzyme secreted by the fly itself. We showed that this serine protease inhibitor (serpin) enzyme, Serpin10, can inactivate triggers of the complement cascade, protect tsetse-infective trypanosomes from complement lysis, and is important for trypanosome establishment in the tsetse midgut. Taken together, we propose that GmmSRPN10 may be part of a repertoire of complement-inhibiting proteins secreted by tsetse that are utilized by T. brucei to evade complement lysis in the tsetse midgut.
PMCID: PMC4287558  PMID: 25569180
2.  Explaining the Host-Finding Behavior of Blood-Sucking Insects: Computerized Simulation of the Effects of Habitat Geometry on Tsetse Fly Movement 
Male and female tsetse flies feed exclusively on vertebrate blood. While doing so they can transmit the diseases of sleeping sickness in humans and nagana in domestic stock. Knowledge of the host-orientated behavior of tsetse is important in designing bait methods of sampling and controlling the flies, and in understanding the epidemiology of the diseases. For this we must explain several puzzling distinctions in the behavior of the different sexes and species of tsetse. For example, why is it that the species occupying savannahs, unlike those of riverine habitats, appear strongly responsive to odor, rely mainly on large hosts, are repelled by humans, and are often shy of alighting on baits?
Methodology/Principal Findings
A deterministic model that simulated fly mobility and host-finding success suggested that the behavioral distinctions between riverine, savannah and forest tsetse are due largely to habitat size and shape, and the extent to which dense bushes limit occupiable space within the habitats. These factors seemed effective primarily because they affect the daily displacement of tsetse, reducing it by up to ∼70%. Sex differences in behavior are explicable by females being larger and more mobile than males.
Habitat geometry and fly size provide a framework that can unify much of the behavior of all sexes and species of tsetse everywhere. The general expectation is that relatively immobile insects in restricted habitats tend to be less responsive to host odors and more catholic in their diet. This has profound implications for the optimization of bait technology for tsetse, mosquitoes, black flies and tabanids, and for the epidemiology of the diseases they transmit.
Author Summary
Tsetse flies and other blood-sucking insects spread devastating diseases of humans and livestock. We must understand the host-finding behavior of these vectors to assess their epidemiological importance and to design optimal bait methods for controlling or sampling them. Unfortunately, mysteries abound in the host-finding behavior of tsetse. For example, it is strange that visual cues are more important for species found in riverine habitats, where dense vegetation restricts the range of visual stimuli, whereas olfactory cues are more important for species occurring in open savannah. To explain this paradox, we used a deterministic model which showed that restricted riverine habitats can reduce tsetse movement by up to ∼70%. This, and the fact that movement increases with fly size, can explain why savannah tsetse, especially the larger ones, rely relatively greatly on olfactory cues, are particularly available to large stationary baits, are repelled by humans, and often investigate baits only briefly without alighting on them. The results also explain why tiny, inexpensive, and odorless baits can control riverine tsetse effectively, whereas larger odor-baited devices are needed against savannah tsetse. These findings have important bearings on the study of host-finding behavior in other blood-sucking insects, including mosquitoes.
PMCID: PMC4055578  PMID: 24921243
3.  An Investigation into the Protein Composition of the Teneral Glossina morsitans morsitans Peritrophic Matrix 
Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), which is an acellular gut lining surrounding the blood meal. Crossing of this multi-layered structure occurs at least twice during parasite migration and development, but the mechanism of how trypanosomes do so is not understood. In order to better comprehend the molecular events surrounding trypanosome penetration of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism.
PMs from male teneral (young, unfed) flies were dissected, solubilised in urea/SDS buffer and the proteins precipitated with cold acetone/TCA. The PM proteins were either subjected to an in-solution tryptic digestion or fractionated on 1D SDS-PAGE, and the resulting bands digested using trypsin. The tryptic fragments from both preparations were purified and analysed by LC-MS/MS.
Overall, nearly 300 proteins were identified from both analyses, several of those containing signature Chitin Binding Domains (CBD), including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, 27 proteins from the tsetse secondary endosymbiont, Sodalis glossinidius, were also identified, suggesting this bacterium is probably in close association with the tsetse PM.
To our knowledge this is the first report on the protein composition of teneral G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology and may help identify potential molecular targets to block trypanosome development within the tsetse.
Author Summary
African trypanosomes are transmitted by the haematophagous tsetse vector. For transmission to occur, bloodmeal ingested trypanosomes must overcome numerous barriers imposed by the fly. The first obstacle is the crossing of peritrophic matrix (PM), a cell-free structure that protects the midgut epithelial cells from coming under attack by the hosts' digestive enzymes, aids in water retention and helps prevent harmful pathogens from establishing a systemic infection. Trypanosomes cross the tsetse PM at least twice in their development but how they do so remains to be elucidated. Despite being a recognised barrier to trypanosome infections, there is limited knowledge of the molecular components of the tsetse PM. In this study we identified nearly 300 PM proteins using two mass spectrometry approaches. Several of the identified components were peritrophins, which are a key group of glycoproteins essential for PM integrity. In addition, we detected proteins from Sodalis glossinidius, a commensal bacterium linked to increased susceptibility to trypanosome infection in tsetse. Our study provides the first comprehensive identification of proteins from the tsetse PM, which provides a starting point for research into potential targets for vector control.
PMCID: PMC3998921  PMID: 24763256
4.  The characterization of the Phlebotomus papatasi transcriptome 
Insect molecular biology  2013;22(2):211-232.
As important vectors of human disease, phlebotomine sand flies are of global significance to human health, transmitting several emerging and re-emerging infectious diseases. The most devastating of the sand fly transmitted infections are the leishmaniases, causing significant mortality and morbidity in both the Old and New World. Here we present the first global transcriptome analysis of the Old World vector of cutaneous leishmaniasis, Phlebotomus papatasi (Scopoli) and compare this transcriptome to that of the New World vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed using pooled mRNA from Phlebotomus papatasi larvae, pupae, adult males and females sugar fed, adult females blood fed and fed blood infected with Leishmania major. A total of 47,615 generated sequences were cleaned and assembled into 17,120 unique transcripts. Of the assembled sequences, 50% (8,837 sequences) were classified using Gene Ontology (GO) terms. This collection of transcripts is comprehensive, as demonstrated by the high number of different GO categories. An in depth analysis has revealed 245 sequences with putative homology to proteins involved in blood and sugar digestion, immune response and peritrophic matrix formation. Twelve of the novel genes, including one trypsin, two peptidoglycan recognition proteins (PGRP) and nine chymotrypsins have a higher expression level during larval stages. Two novel chymotrypsins and one novel PGRP are abundantly expressed upon blood feeding. This study will greatly improve the available genomic resources for Ph. papatasi and will provide essential information for annotation of the full genome.
PMCID: PMC3594503  PMID: 23398403
5.  Community Acceptance of Tsetse Control Baits: A Qualitative Study in Arua District, North West Uganda 
There is renewed vigour in efforts to eliminate neglected tropical diseases including sleeping sickness (human African trypanosomiasis or HAT), including attempts to develop more cost-effective methods of tsetse control. In the West Nile region of Uganda, newly designed insecticide-treated targets are being deployed over an area of ∼500 km2. The operational area covers villages where tsetse control has not been conducted previously. The effectiveness of the targets will depend, in part, on their acceptance by the local community.
Methodology/Principal Findings
We assessed knowledge, perceptions and acceptance of tsetse baits (traps, targets) in villages where they had or had not been used previously. We conducted sixteen focus group discussions with male and female participants in eight villages across Arua District. Discussions were audio recorded, translated and transcribed. We used thematic analysis to compare the views of both groups and identify salient themes.
Despite the villages being less than 10 km apart, community members perceived deployed baits very differently. Villagers who had never seen traps before expressed fear, anxiety and panic when they first encountered them. This was related to associations with witchcraft and “ghosts from the river” which are traditionally linked with physical or mental illness, death and misfortune. By contrast, villagers living in areas where traps had been used previously had positive attitudes towards them and were fully aware of their purpose and benefits. The latter group reported that they had similar negative perceptions when tsetse control interventions first started a decade ago. Our results suggest that despite their proximity, acceptance of traps varies markedly between villages and this is related to the duration of experience with tsetse control programs. The success of community-based interventions against tsetse will therefore depend on early engagements with communities and carefully designed sensitization campaigns that reach all communities, especially those living in areas new to such interventions.
Author Summary
Sleeping sickness is a disease which results in serious physical and mental symptoms and is ultimately deadly if not treated. It is caused by sub-species of Trypanosoma brucei transmitted by tsetse which live exclusively in Africa. Currently, the only preventive measure against sleeping sickness is reduction of tsetse population in the areas where these flies and humans share the same living space. This can be achieved through the use of traps or insecticide-treated targets to attract and kill tsetse. As the traps are newly introduced in some areas, we explored how local communities perceive them. We compared their views to those of communities living in areas where traps have been used sporadically for more than 10 years. Despite villages with or without experience of vector control being less than 10 km apart, they had very different perceptions: the group new to targets had many negative perceptions, associated with witchcraft and supernatural powers, while the group knowing targets from the past perceived them positively and beneficial. Understanding of local perceptions is important, because it will help us to involve communities affected by sleeping sickness in tsetse control programs. Without their support these programs are short-lived and ineffective.
PMCID: PMC3861179  PMID: 24349593
7.  Control Using Genetically Modified Insects Poses Problems for Regulators 
PMCID: PMC3269408  PMID: 22303494
8.  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.
PMCID: PMC3210762  PMID: 22087240
9.  Vegetation and the Importance of Insecticide-Treated Target Siting for Control of Glossina fuscipes fuscipes 
Control of tsetse flies using insecticide-treated targets is often hampered by vegetation re-growth and encroachment which obscures a target and renders it less effective. Potentially this is of particular concern for the newly developed small targets (0.25 high × 0.5 m wide) which show promise for cost-efficient control of Palpalis group tsetse flies. Consequently the performance of a small target was investigated for Glossina fuscipes fuscipes in Kenya, when the target was obscured following the placement of vegetation to simulate various degrees of natural bush encroachment. Catches decreased significantly only when the target was obscured by more than 80%. Even if a small target is underneath a very low overhanging bush (0.5 m above ground), the numbers of G. f. fuscipes decreased by only about 30% compared to a target in the open. We show that the efficiency of the small targets, even in small (1 m diameter) clearings, is largely uncompromised by vegetation re-growth because G. f. fuscipes readily enter between and under vegetation. The essential characteristic is that there should be some openings between vegetation.
This implies that for this important vector of HAT, and possibly other Palpalis group flies, a smaller initial clearance zone around targets can be made and longer interval between site maintenance visits is possible both of which will result in cost savings for large scale operations. We also investigated and discuss other site features e.g. large solid objects and position in relation to the water's edge in terms of the efficacy of the small targets.
Author Summary
Sleeping Sickness (Human African Trypanosomiasis) is a serious threat to health and development in sub-Saharan Africa. Due to lack of vaccines and prophylactic drugs, vector control is the only method of disease prevention. Small (0.25×0.5 m) insecticide-treated targets have been shown to be cost-efficient for several Palpalis group tsetse flies, but there are concerns that they may become obscured by vegetation with a subsequent reduction in efficiency. We showed that the efficiency of the small targets was largely uncompromised by vegetation encroachment because G. f. fuscipes readily enter between and under vegetation to locate a small target, e.g. into small (1 m diameter) site clearings and underneath a very low (0.5 m) canopy. This implies that the dense vegetation, typical of the riverine habitats of Palpalis group tsetse, will not compromise the performance of tiny targets, as long as there are adequate openings of >30 cm between vegetation. Moreover, the maintanence of cleared areas around targets seems less important for the control of G. f. fuscipes with consequent savings in costs for control operations.
PMCID: PMC3176746  PMID: 21949897
10.  Towards an Optimal Design of Target for Tsetse Control: Comparisons of Novel Targets for the Control of Palpalis Group Tsetse in West Africa 
Tsetse flies of the Palpalis group are the main vectors of sleeping sickness in Africa. Insecticide impregnated targets are one of the most effective tools for control. However, the cost of these devices still represents a constraint to their wider use. The objective was therefore to improve the cost effectiveness of currently used devices.
Methodology/Principal Findings
Experiments were performed on three tsetse species, namely Glossina palpalis gambiensis and G. tachinoides in Burkina Faso and G. p. palpalis in Côte d'Ivoire. The 1×1 m2 black blue black target commonly used in W. Africa was used as the standard, and effects of changes in target size, shape, and the use of netting instead of black cloth were measured. Regarding overall target shape, we observed that horizontal targets (i.e. wider than they were high) killed 1.6-5x more G. p. gambiensis and G. tachinoides than vertical ones (i.e. higher than they were wide) (P<0.001). For the three tsetse species including G. p. palpalis, catches were highly correlated with the size of the target. However, beyond the size of 0.75 m, there was no increase in catches. Replacing the black cloth of the target by netting was the most cost efficient for all three species.
Reducing the size of the current 1*1 m black-blue-black target to horizontal designs of around 50 cm and replacing black cloth by netting will improve cost effectiveness six-fold for both G. p. gambiensis and G. tachinoides. Studying the visual responses of tsetse to different designs of target has allowed us to design more cost-effective devices for the effective control of sleeping sickness and animal trypanosomiasis in Africa.
Author Summary
Tsetse flies transmit trypanosomes causing sleeping sickness and nagana. Controlling tsetse prevents transmission of these diseases. Insecticide impregnated targets are highly effective but are too costly. This study aims to improve the cost effectiveness of targets. Experiments were performed on three tsetse species in Burkina Faso or Côte d'Ivoire. Effects of target size, shape, and the use of netting instead of black cloth were measured. We observed that targets wider than they are high (horizontal target) killed 1.6-5x more G. p. gambiensis and G. tachinoides than vertical ones. Catches were highly correlated with the size of the target up to a target size of 0.75 m, beyond which there was no further increase in catches. Replacing the black cloth of the target by netting did not change catches, but was far cheaper. Hence reducing the size of the current 1 m×1 m black-blue-black target to an horizontal 0.75×0.5 m net blue net target will improve cost effectiveness six-fold for both G. p. gambiensis and G. tachinoides. Studying the visual responses of tsetse to different designs of target has allowed us to design more cost-effective devices for the effective control of sleeping sickness and animal trypanosomiasis in Africa.
PMCID: PMC3176748  PMID: 21949896
11.  Cryptic Diversity within the Major Trypanosomiasis Vector Glossina fuscipes Revealed by Molecular Markers 
The tsetse fly Glossina fuscipes s.l. is responsible for the transmission of approximately 90% of cases of human African trypanosomiasis (HAT) or sleeping sickness. Three G. fuscipes subspecies have been described, primarily based upon subtle differences in the morphology of their genitalia. Here we describe a study conducted across the range of this important vector to determine whether molecular evidence generated from nuclear DNA (microsatellites and gene sequence information), mitochondrial DNA and symbiont DNA support the existence of these taxa as discrete taxonomic units.
Principal Findings
The nuclear ribosomal Internal transcribed spacer 1 (ITS1) provided support for the three subspecies. However nuclear and mitochondrial sequence data did not support the monophyly of the morphological subspecies G. f. fuscipes or G. f. quanzensis. Instead, the most strongly supported monophyletic group was comprised of flies sampled from Ethiopia. Maternally inherited loci (mtDNA and symbiont) also suggested monophyly of a group from Lake Victoria basin and Tanzania, but this group was not supported by nuclear loci, suggesting different histories of these markers. Microsatellite data confirmed strong structuring across the range of G. fuscipes s.l., and was useful for deriving the interrelationship of closely related populations.
We propose that the morphological classification alone is not used to classify populations of G. fuscipes for control purposes. The Ethiopian population, which is scheduled to be the target of a sterile insect release (SIT) programme, was notably discrete. From a programmatic perspective this may be both positive, given that it may reflect limited migration into the area or negative if the high levels of differentiation are also reflected in reproductive isolation between this population and the flies to be used in the release programme.
Author Summary
Glossina fuscipes s.l. tsetse flies are responsible for transmission of approximately 90% of the cases of Human African Typanosomiasis in Sub Saharan Africa. It was previously proposed on the basis of morphology that G. fuscipes is composed of three sub-species. Using genetic evidence from G. fuscipes nuclear, mitochondrial and symbiont DNA, we show that the morphological subspecies do not correspond well to genetic differences between the flies and morphologically similar flies may have arisen more than once in the evolution of this species. Instead, we found at least 5 main allopatrically distributed groups of G. fuscipes flies. The most genetically distinct group of flies originated from Ethiopia, where a sterile insect release programme is planned. Given that tsetse control often exploits species-specific behaviours there is a pressing need to establish the taxonomic status and ranges of these five groups. Moreover given that we were only able to perform limited sampling in many parts of the species distribution further groups within G. fuscipes are likely to be awaiting discovery.
PMCID: PMC3153427  PMID: 21858237
12.  How Do Tsetse Recognise Their Hosts? The Role of Shape in the Responses of Tsetse (Glossina fuscipes and G. palpalis) to Artificial Hosts 
Palpalis-group tsetse, particularly the subspecies of Glossina palpalis and G. fuscipes, are the most important transmitters of human African trypanomiasis (HAT), transmitting >95% of cases. Traps and insecticide-treated targets are used to control tsetse but more cost-effective baits might be developed through a better understanding of the fly's host-seeking behaviour. Electrocuting grids were used to assess the numbers of G. palpalis palpalis and G. fuscipes quanzensis attracted to and landing on square or oblong targets of black cloth varying in size from 0.01 m2 to 1.0 m2. For both species, increasing the size of a square target from 0.01 m2 (dimensions = 0.1×0.1 m) to 1.0 m2 (1.0×1.0 m) increased the catch ∼4x however the numbers of tsetse killed per unit area of target declined with target size suggesting that the most cost efficient targets are not the largest. For G. f. quanzensis, horizontal oblongs, (1 m wide×0.5 m high) caught ∼1.8x more tsetse than vertical ones (0.5 m wide×1.0 m high) but the opposite applied for G. p. palpalis. Shape preference was consistent over the range of target sizes. For G. p. palpalis square targets caught as many tsetse as the oblong; while the evidence is less strong the same appears to apply to G. f. quanzensis. The results suggest that targets used to control G. p. palpalis and G. f. quanzensis should be square, and that the most cost-effective designs, as judged by the numbers of tsetse caught per area of target, are likely to be in the region of 0.25×0.25 m2. The preference of G. p. palpalis for vertical oblongs is unique amongst tsetse species, and it is suggested that this response might be related to its anthropophagic behaviour and hence importance as a vector of HAT.
Author Summary
While the numbers of cases of human African trypanosomiasis (HAT) is now less than 10,000 reported cases per year, progress against the tsetse species that spread the disease is poor, with ∼10 million square kilometres of sub-Saharan Africa still being infested. This widespread persistence of vectors and reservoir hosts threatens the long-term sustainability of recent gains against HAT. Better progress against the vector would be achieved by developing cheap, effective and practical methods of tsetse control. Toward this end, we are improving the design of insecticide-treated targets to attract and kill tsetse. Here we show that for two important vectors of HAT, Glossina palpalis palpalis in Côte d'Ivoire and Glossina fuscipes quanzensis in the Democratic Republic of Congo, small (between 0.25 m and 0.5 m square) targets of black cloth with equally sized panel of fine black netting are ∼10x more cost-effective than the larger (∼1 m square) targets or traps commonly in use.
PMCID: PMC3149008  PMID: 21829734
13.  Improving the Cost-Effectiveness of Visual Devices for the Control of Riverine Tsetse Flies, the Major Vectors of Human African Trypanosomiasis 
Control of the Riverine (Palpalis) group of tsetse flies is normally achieved with stationary artificial devices such as traps or insecticide-treated targets. The efficiency of biconical traps (the standard control device), 1×1 m black targets and small 25×25 cm targets with flanking nets was compared using electrocuting sampling methods. The work was done on Glossina tachinoides and G. palpalis gambiensis (Burkina Faso), G. fuscipes quanzensis (Democratic Republic of Congo), G. f. martinii (Tanzania) and G. f. fuscipes (Kenya). The killing effectiveness (measured as the catch per m2 of cloth) for small targets plus flanking nets is 5.5–15X greater than for 1 m2 targets and 8.6–37.5X greater than for biconical traps. This has important implications for the costs of control of the Riverine group of tsetse vectors of sleeping sickness.
Author Summary
Sleeping Sickness (Human African Trypanosomiasis) is a serious threat to health and development in sub-Saharan Africa. Currently there are no vaccines or prophylactic drugs available to prevent contraction of the disease. Consequently vector control is the only method of disease prevention. In many areas, especially those lacking high densities of cattle, the only control option for routine use against tsetse flies are insecticide-treated targets or biconical traps. However, these methods in their current form are often too expensive for routine use against the riverine tsetse species that are the major vectors of sleeping sickness. Our aim is to develop a more cost-effective device than those currently available. Working on four species of tsetse fly we have shown that a small 25×25 cm target with adjacent flanking net was up to 38x more cost-effective at killing tsetse flies than existing devices. These findings suggest that this new technology may make vector control in HAT foci an affordable option.
PMCID: PMC3149014  PMID: 21829743
14.  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
15.  Tsetse EP Protein Protects the Fly Midgut from Trypanosome Establishment 
PLoS Pathogens  2010;6(3):e1000793.
African trypanosomes undergo a complex developmental process in their tsetse fly vector before transmission back to a vertebrate host. Typically, 90% of fly infections fail, most during initial establishment of the parasite in the fly midgut. The specific mechanism(s) underpinning this failure are unknown. We have previously shown that a Glossina-specific, immunoresponsive molecule, tsetse EP protein, is up regulated by the fly in response to gram-negative microbial challenge. Here we show by knockdown using RNA interference that this tsetse EP protein acts as a powerful antagonist of establishment in the fly midgut for both Trypanosoma brucei brucei and T. congolense. We demonstrate that this phenomenon exists in two species of tsetse, Glossina morsitans morsitans and G. palpalis palpalis, suggesting tsetse EP protein may be a major determinant of vector competence in all Glossina species. Tsetse EP protein levels also decline in response to starvation of the fly, providing a possible explanation for increased susceptibility of starved flies to trypanosome infection. As starvation is a common field event, this fact may be of considerable importance in the epidemiology of African trypanosomiasis.
Author Summary
In Africa, tsetse flies transmit the trypanosomes causing the devastating diseases sleeping sickness in man and nagana in domesticated animals. These diseases are major causes of underdevelopment in Africa. Paradoxically, most, but not all, flies are resistant to infection with trypanosomes, but we do not have a clear picture of how flies fight off trypanosomes. Here we show that a particular, tsetse-specific immune responsive protein called tsetse EP acts as a powerful antagonist of trypanosome establishment in the fly midgut. It is known that starvation of flies leads to an increase in their susceptibility to trypanosomes and this may be a considerable factor in the epidemiology of the disease in Africa. Here we demonstrate that starvation leads to a decrease in tsetse EP levels, which may explain how starvation of the fly works to increase its susceptibility.
PMCID: PMC2832768  PMID: 20221444
16.  The sialotranscriptome of the blood-sucking bug Triatoma brasiliensis (Hemiptera, Triatominae) 
Triatoma brasiliensis is the most important autochthon vector of Trypanosoma cruzi in Brazil, where it is widely distributed in the semiarid areas of the Northeast. In order to advance the knowledge of the salivary biomolecules of Triatominae, a salivary gland cDNA library of T. brasiliensis was mass sequenced and analyzed. Polypeptides were sequenced by HPLC/Edman degradation experiments. 1,712 cDNA sequences were obtained and grouped in 786 clusters. The housekeeping category had 24.4% and 17.8% of the clusters and sequences, respectively. The putatively secreted category contained 47.1% of the clusters and 68.2% of the sequences. Finally, 28.5% of the clusters, containing 14% of all sequences, were classified as unknown. The sialoma of T. brasiliensis showed a high amount and great variety of different lipocalins (93.8% of secreted proteins). Remarkably, a great number of serine proteases that were not observed in previous blood-sucking sialotranscriptomes were found. Nine Kazal peptides were identified, among them one with high homology to the tabanid vasodilator vasotab, suggesting that the Triatoma vasodilator could be a Kazal protein.
PMCID: PMC1896098  PMID: 17550826
Saliva; Transcriptome; Hematophagy; Salivary proteins; Triatoma brasiliensis

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