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.

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.

Background

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.

Conclusion/Significance

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.

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.

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.

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.

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.

Background

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.

Conclusion/Significance

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.

Background

Giardia duodenalis is prevalent in tropical settings where diverse opportunities exist for transmission between people and animals. We conducted a cross-sectional study of G. duodenalis in people, livestock, and wild primates near Kibale National Park, Uganda, where human-livestock-wildlife interaction is high due to habitat disturbance. Our goal was to infer the cross-species transmission potential of G. duodenalis using molecular methods and to investigate clinical consequences of infection.

Methodology/Principal Findings

Real-time PCR on DNA extracted from fecal samples revealed a combined prevalence of G. duodenalis in people from three villages of 44/108 (40.7%), with prevalence reaching 67.5% in one village. Prevalence rates in livestock and primates were 12.4% and 11.1%, respectively. Age was associated with G. duodenalis infection in people (higher prevalence in individuals ≤15 years) and livestock (higher prevalence in subadult versus adult animals), but other potential risk factors in people (gender, contact with domestic animals, working in fields, working in forests, source of drinking water, and medication use) were not. G. duodenalis infection was not associated with gastrointestinal symptoms in people, nor was clinical disease noted in livestock or primates. Sequence analysis of four G. duodenalis genes identified assemblage AII in humans, assemblage BIV in humans and endangered red colobus monkeys, and assemblage E in livestock and red colobus, representing the first documentation of assemblage E in a non-human primate. In addition, genetic relationships within the BIV assemblage revealed sub-clades of identical G. duodenalis sequences from humans and red colobus.

Conclusions/Significance

Our finding of G. duodenalis in people and primates (assemblage BIV) and livestock and primates (assemblage E) underscores that cross-species transmission of multiple G. duodenalis assemblages may occur in locations such as western Uganda where people, livestock, and primates overlap in their use of habitat. Our data also demonstrate a high but locally variable prevalence of G. duodenalis in people from western Uganda, but little evidence of associated clinical disease. Reverse zoonotic G. duodenalis transmission may be particularly frequent in tropical settings where anthropogenic habitat disturbance forces people and livestock to interact at high rates with wildlife, and this could have negative consequences for wildlife conservation.

Author Summary

Giardia duodenalis is a common protozoan parasite that infects multiple mammalian species, including humans. We analyzed G. duodenalis from people, livestock, and wild non-human primates in forest fragments near Kibale National Park, western Uganda, where habitat disturbance and human-animal interaction are high. Molecular analyses indicated that endangered red colobus monkeys were infected with G. duodenalis assemblages BIV and E, which characteristically infect humans and livestock, respectively. G. duodenalis infected people at rates of up to 67.5% in one village, and people age 15 years or younger were especially likely to be infected. G. duodenalis infection in people was not associated with other factors related to behavior and hygiene, and infected people were no more likely to have reported gastrointestinal symptoms than were uninfected people. These results demonstrate that G. duodenalis transmission from humans and domestic animals to wildlife may occur with ease in locations such as western Uganda, where habitat disturbance causes ecological overlap among people, livestock, and primates. This conclusion has conservation implications for wildlife such as red colobus, which are already endangered by habitat loss.

Background

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.

Results

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.

Conclusions

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.