Structure of some East African Glossina fuscipes fuscipes populations

Abstract.  Glossina fuscipes fuscipes Newstead 1910 (Diptera: Glossinidae) is the primary vector of human sleeping sickness in Kenya and Uganda. This is the first report on its population structure. A total of 688 nucleotides of mitochondrial ribosomal 16S2 and cytochrome oxidase I genes were sequenced. Twenty-one variants were scored in 79 flies from three geographically diverse natural populations. Four haplotypes were shared among populations, eight were private and nine were singletons. The mean haplotype and nucleotide diversities were 0.84 and 0.009, respectively. All populations were genetically differentiated and were at demographic equilibrium. In addition, a longstanding laboratory culture originating from the Central African Republic (CAR-lab) in 1986 (or before) was examined. Haplotype and nucleotide diversities in this culture were 0.95 and 0.012, respectively. None of its 27 haplotypes were shared with the East African populations. A first approximation of relative effective population sizes was Uganda > CAR-lab > Kenya. It was concluded that the structure of G. f. fuscipes populations in East Africa is localized.     

Prospects of a fungus-contamination device for the control of tsetse fly Glossina fuscipes fuscipes

The prospects of the fungus Metarhizium anisopliae (Metsch.) Sorok. applied in contamination devices (Cds) to control tsetse fly Glossina fuscipes fuscipes Newstead was tested in a field experiment in Lake Victoria from 2 March 1999 to 31 August 2000. One hundred and sixty pyramidal traps mounted with Cds were deployed along the lakeshore and rivers on Mfangano Island. Contamination devices were loaded with 1.5-2.0 g of dry conidia/Cd. On the second island, Nzenze Island, four pyramidal traps fitted with plastic bags were deployed and served as the conventional 'trap and kill' population suppression method. A third island, Ngodhe Island, remained untreated and served as a control. Cds were recharged monthly with fresh conidia; plastic bags were also changed monthly. The apparent changes in population density were monitored weekly using biconical traps set at random on the three islands. To assess the incidence of M. anisopliae in tsetse flies on Mfangano Island, flies captured during monitoring were maintained in the laboratory and their mortality recorded. Fly population was reduced to 82.4 and 95.8% relative to untreated control on Mfangano and Nzenze islands, respectively, during the experimental period. Compared to the fungus-treated island, the number of flies caught in monitoring traps increased considerably in 'trap kill' treatment at 5 months after the treatments were removed. The incidence of M. anisopliae in fly populations was low during the 12 weeks following the initiation of the experiment but increased afterward until termination of the treatment. M. anisopliae could still be recovered from fly populations at 3 months after termination of the treatment, although the incidence was low. The results of this study have shown that application of M. anisopliae in a contamination device can suppress the population of G. fuscipes fuscipes comparable to the 'trap and kill' technology.     

Implications of microfauna-host interactions for trypanosome transmission dynamics in Glossina fuscipes fuscipes in Uganda

Tsetse flies (Diptera: Glossinidae) are vectors for African trypanosomes (Euglenozoa: kinetoplastida), protozoan parasites that cause African trypanosomiasis in humans (HAT) and nagana in livestock. In addition to trypanosomes, two symbiotic bacteria (Wigglesworthia glossinidia and Sodalis glossinidius) and two parasitic microbes, Wolbachia and a salivary gland hypertrophy virus (SGHV), have been described in tsetse. Here we determined the prevalence of and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in Glossina fuscipes fuscipes in Uganda over a large geographical scale spanning the range of host genetic and spatial diversity. Using a multivariate analysis approach, we uncovered complex coinfection dynamics between the pathogens and statistically significant associations between host genetic groups and pathogen prevalence. It is important to note that these coinfection dynamics and associations with the host were not apparent by univariate analysis. These associations between host genotype and pathogen are particularly evident for Wolbachia and SGHV where host groups are inversely correlated for Wolbachia and SGHV prevalence. On the other hand, trypanosome infection prevalence is more complex and covaries with the presence of the other two pathogens, highlighting the importance of examining multiple pathogens simultaneously before making generalizations about infection and spatial patterns. It is imperative to note that these novel findings would have been missed if we had employed the standard univariate analysis used in previous studies. Our results are discussed in the context of disease epidemiology and vector control.     

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.     

Infection with endosymbiotic Spiroplasma disrupts tsetse (Glossina fuscipes fuscipes) metabolic and reproductive homeostasis

Tsetse flies (Glossina spp.) house a population-dependent assortment of microorganisms that can include pathogenic African trypanosomes and maternally transmitted endosymbiotic bacteria, the latter of which mediate numerous aspects of their host’s metabolic, reproductive, and immune physiologies. One of these endosymbionts, Spiroplasma, was recently discovered to reside within multiple tissues of field captured and laboratory colonized tsetse flies grouped in the Palpalis subgenera. In various arthropods, Spiroplasma induces reproductive abnormalities and pathogen protective phenotypes. In tsetse, Spiroplasma infections also induce a protective phenotype by enhancing the fly’s resistance to infection with trypanosomes. However, the potential impact of Spiroplasma on tsetse’s viviparous reproductive physiology remains unknown. Herein we employed high-throughput RNA sequencing and laboratory-based functional assays to better characterize the association between Spiroplasma and the metabolic and reproductive physiologies of G. fuscipes fuscipes (Gff), a prominent vector of human disease. Using field-captured Gff, we discovered that Spiroplasma infection induces changes of sex-biased gene expression in reproductive tissues that may be critical for tsetse’s reproductive fitness. Using a Gff lab line composed of individuals heterogeneously infected with Spiroplasma, we observed that the bacterium and tsetse host compete for finite nutrients, which negatively impact female fecundity by increasing the length of intrauterine larval development. Additionally, we found that when males are infected with Spiroplasma, the motility of their sperm is compromised following transfer to the female spermatheca. As such, Spiroplasma infections appear to adversely impact male reproductive fitness by decreasing the competitiveness of their sperm. Finally, we determined that the bacterium is maternally transmitted to intrauterine larva at a high frequency, while paternal transmission was also noted in a small number of matings. Taken together, our findings indicate that Spiroplasma exerts a negative impact on tsetse fecundity, an outcome that could be exploited for reducing tsetse population size and thus disease transmission.     

High levels of genetic differentiation between Ugandan Glossina fuscipes fuscipes populations separated by Lake Kyoga

Background

Glossina fuscipes fuscipes is the major vector of human African trypanosomiasis, commonly referred to as sleeping sickness, in Uganda. In western and eastern Africa, the disease has distinct clinical manifestations and is caused by two different parasites: Trypanosoma brucei rhodesiense and T. b. gambiense. Uganda is exceptional in that it harbors both parasites, which are separated by a narrow 160-km belt. This separation is puzzling considering there are no restrictions on the movement of people and animals across this region.

Methodology and Results

We investigated whether genetic heterogeneity of G. f. fuscipes vector populations can provide an explanation for this disjunct distribution of the Trypanosoma parasites. Therefore, we examined genetic structuring of G. f. fuscipes populations across Uganda using newly developed microsatellite markers, as well as mtDNA. Our data show that G. f. fuscipes populations are highly structured, with two clearly defined clusters that are separated by Lake Kyoga, located in central Uganda. Interestingly, we did not find a correlation between genetic heterogeneity and the type of Trypanosoma parasite transmitted.

Conclusions

The lack of a correlation between genetic structuring of G. f. fuscipes populations and the distribution of T. b. gambiense and T. b. rhodesiense indicates that it is unlikely that genetic heterogeneity of G. f. fuscipes populations explains the disjunct distribution of the parasites. These results have important epidemiological implications, suggesting that a fusion of the two disease distributions is unlikely to be prevented by an incompatibility between vector populations and parasite.     

The population genomics of multiple tsetse fly (Glossina fuscipes fuscipes) admixture zones in Uganda

Understanding the mechanisms that enforce, maintain or reverse the process of speciation is an important challenge in evolutionary biology. This study investigates the patterns of divergence and discusses the processes that form and maintain divergent lineages of the tsetse fly Glossina fuscipes fuscipes in Uganda. We sampled 251 flies from 18 sites spanning known genetic lineages and the four admixture zones between them. We apply population genomics, hybrid zone and approximate Bayesian computation to the analysis of three types of genetic markers: 55,267 double‐digest restriction site‐associated DNA (ddRAD) SNPs to assess genome‐wide admixture, 16 microsatellites to provide continuity with published data and accurate biogeographic modelling, and a 491‐bp fragment of mitochondrial cytochrome oxidase I and II to infer maternal inheritance patterns. Admixture zones correspond with regions impacted by the reorganization of Uganda's river networks that occurred during the formation of the West African Rift system over the last several hundred thousand years. Because tsetse fly population distributions are defined by rivers, admixture zones likely represent both old and new regions of secondary contact. Our results indicate that older hybrid zones contain mostly parental types, while younger zones contain variable hybrid types resulting from multiple generations of interbreeding. These findings suggest that reproductive barriers are nearly complete in the older admixture zones, while nearly absent in the younger admixture zones. Findings are consistent with predictions of hybrid zone theory: Populations in zones of secondary contact transition rapidly from early to late stages of speciation or collapse all together.     

The interaction of taste and heat on the biting response of the tsetse fly Glossina fuscipes fuscipes

Abstract.Tsetse flies probe more on a heated surface with a trace of uric acid than on one without. Uric acid is one of the components of human sweat and it elicits spike responses from taste hairs on the flies' legs. In this paper it is examined how heat from the surface and taste interact to affect the biting behaviour of Glossina fuscipes fuscipes Newstead 1910 (Diptera: Glossinidae) over successive days of food deprivation. The biting behaviour consists of bouts of probing, both ambulatory and stationary, intercalated with short hops of flight. The number of bouts increases over successive days, whereas the average bout duration does not. Although uric acid alone could not induce the flies to probe, in combination with surface heat it affected the flies greatly. Average bout duration was two‐fold that on a heated surface not treated with uric acid. The frequency of bouts was not affected by uric acid. These experiments and auxiliary ones on mechanoreceptive input and odours lead to the insight that the factors which affect biting behaviour can be viewed as a hierarchy. The hierarchy extends from those that affect the onset of biting to those that affect its course.     

Optimizing the colour and fabric of targets for the control of the tsetse fly Glossina fuscipes fuscipes

Background

Most cases of human African trypanosomiasis (HAT) start with a bite from one of the subspecies of Glossina fuscipes. Tsetse use a range of olfactory and visual stimuli to locate their hosts and this response can be exploited to lure tsetse to insecticide-treated targets thereby reducing transmission. To provide a rational basis for cost-effective designs of target, we undertook studies to identify the optimal target colour.

Methodology/Principal Findings

On the Chamaunga islands of Lake Victoria , Kenya, studies were made of the numbers of G. fuscipes fuscipes attracted to targets consisting of a panel (25 cm square) of various coloured fabrics flanked by a panel (also 25 cm square) of fine black netting. Both panels were covered with an electrocuting grid to catch tsetse as they contacted the target. The reflectances of the 37 different-coloured cloth panels utilised in the study were measured spectrophotometrically. Catch was positively correlated with percentage reflectance at the blue (460 nm) wavelength and negatively correlated with reflectance at UV (360 nm) and green (520 nm) wavelengths. The best target was subjectively blue, with percentage reflectances of 3%, 29%, and 20% at 360 nm, 460 nm and 520 nm respectively. The worst target was also, subjectively, blue, but with high reflectances at UV (35% reflectance at 360 nm) wavelengths as well as blue (36% reflectance at 460 nm); the best low UV-reflecting blue caught 3× more tsetse than the high UV-reflecting blue.

Conclusions/Significance

Insecticide-treated targets to control G. f. fuscipes should be blue with low reflectance in both the UV and green bands of the spectrum. Targets that are subjectively blue will perform poorly if they also reflect UV strongly. The selection of fabrics for targets should be guided by spectral analysis of the cloth across both the spectrum visible to humans and the UV region.     

Effects of vegetation and weather on trap catches of Glossina fuscipes fuscipes near Lake Victoria, Kenya

Glossina fuscipes fuscipes Newstead was sampled in isolated thickets and forest patches near Lake Victoria, Kenya using unbaited biconical traps, between March 1992 and June 1993. Traps set at 1 m from the forest edge caught 3.3 times as many males and 5 times as many females as those set inside or 10 m away. The corresponding figures at 1 m from the edge of thicket were about 1.43 and 1.64 times, respectively. Hourly catches of males and females were positively correlated with temperature, light intensity and host (monitor lizard) prevalence, and negatively correlated with relative humidity. Light intensity and temperature were the most important variables affecting the catches of each sex. The results are discussed in relation to control and monitoring of G. f. fuscipes using traps.     

Phylogeography and Population Structure of Glossina fuscipes fuscipes in Uganda: Implications for Control of Tsetse

Background

Glossina fuscipes fuscipes, a riverine species of tsetse, is the main vector of both human and animal trypanosomiasis in Uganda. Successful implementation of vector control will require establishing an appropriate geographical scale for these activities. Population genetics can help to resolve this issue by characterizing the extent of linkage among apparently isolated groups of tsetse.

Methodology/Principal Findings

We conducted genetic analyses on mitochondrial and microsatellite data accumulated from approximately 1000 individual tsetse captured in Uganda and neighboring regions of Kenya and Sudan. Phylogeographic analyses suggested that the largest scale genetic structure in G. f. fuscipes arose from an historical event that divided two divergent mitochondrial lineages. These lineages are currently partitioned to northern and southern Uganda and co-occur only in a narrow zone of contact extending across central Uganda. Bayesian assignment tests, which provided evidence for admixture between northern and southern flies at the zone of contact and evidence for northerly gene flow across the zone of contact, indicated that this structure may be impermanent. On the other hand, microsatellite structure within the southern lineage indicated that gene flow is currently limited between populations in western and southeastern Uganda. Within regions, the average F_ST between populations separated by less than 100 km was less than ∼0.1. Significant tests of isolation by distance suggested that gene flow is ongoing between neighboring populations and that island populations are not uniformly more isolated than mainland populations.

Conclusions/Significance

Despite the presence of population structure arising from historical colonization events, our results have revealed strong signals of current gene flow within regions that should be accounted for when planning tsetse control in Uganda. Populations in southeastern Uganda appeared to receive little gene flow from populations in western or northern Uganda, supporting the feasibility of area wide control in the Lake Victoria region by the Pan African Tsetse and Trypanosomiasis Eradication Campaign.     

A spatial genetics approach to inform vector control of tsetse flies (Glossina fuscipes fuscipes) in Northern Uganda

Tsetse flies (genus Glossina) are the only vector for the parasitic trypanosomes responsible for sleeping sickness and nagana across sub‐Saharan Africa. In Uganda, the tsetse fly Glossina fuscipes fuscipes is responsible for transmission of the parasite in 90% of sleeping sickness cases, and co‐occurrence of both forms of human‐infective trypanosomes makes vector control a priority. We use population genetic data from 38 samples from northern Uganda in a novel methodological pipeline that integrates genetic data, remotely sensed environmental data, and hundreds of field‐survey observations. This methodological pipeline identifies isolated habitat by first identifying environmental parameters correlated with genetic differentiation, second, predicting spatial connectivity using field‐survey observations and the most predictive environmental parameter(s), and third, overlaying the connectivity surface onto a habitat suitability map. Results from this pipeline indicated that net photosynthesis was the strongest predictor of genetic differentiation in G. f. fuscipes in northern Uganda. The resulting connectivity surface identified a large area of well‐connected habitat in northwestern Uganda, and twenty‐four isolated patches on the northeastern margin of the G. f. fuscipes distribution. We tested this novel methodological pipeline by completing an ad hoc sample and genetic screen of G. f. fuscipes samples from a model‐predicted isolated patch, and evaluated whether the ad hoc sample was in fact as genetically isolated as predicted. Results indicated that genetic isolation of the ad hoc sample was as genetically isolated as predicted, with differentiation well above estimates made in samples from within well‐connected habitat separated by similar geographic distances. This work has important practical implications for the control of tsetse and other disease vectors, because it provides a way to identify isolated populations where it will be safer and easier to implement vector control and that should be prioritized as study sites during the development and improvement of vector control methods.     

Standardizing Visual Control Devices for Tsetse Flies: East African Species Glossina fuscipes fuscipes and Glossina tachinoides

BackgroundRiverine species of tsetse are responsible for most human African trypanosomiasis (HAT) transmission and are also important vectors of animal trypanosomiasis. This study concerns the development of visual control devices for two such species, Glossina fuscipes fuscipes and Glossina tachinoides, at the eastern limits of their continental range. The goal was to determine the most long-lasting, practical and cost-effective visually attractive device that induces the strongest landing responses in these species for use as insecticide-impregnated tools in vector population suppression.Conclusions/SignificanceTaking into account practical considerations and fly preferences for edges and colours, we propose a 0.5×0.75 m blue-black target as a simple cost-effective device for management of G. f. fuscipes and G. tachinoides, impregnated with insecticide for control and covered with adhesive film for population sampling.     

Estimation of the efficiency of the biconical trap for Glossina fuscipes fuscipes along the lake Victoria shore, Kenya

Prior to estimating the efficiency of the unbaited biconical trap for Glossina fuscipes fuscipes Newstead (Diptera:Glossinidae) the flying height of the insects was estimated using 1 m2 electrified nets placed at 0 and 4 m above the ground level. The degree of avoidance of these nets by the flies was determined by comparing catches in traps surrounded and those not surrounded by incomplete rings of nets. On the basis of the catches in traps surrounded by nets, the expected catches on both sides of the nets were computed and compared with the observed catches, to further estimate this degree of avoidance. About 48% of males and 35% of females were captured above one metre. An average of 61% of males and 40% of females appeared to avoid the nets. Between 18% and 40% fewer tsetse were caught in traps surrounded by an incomplete ring of nets of respectively 1 m and 2 m radius than in traps not surrounded. After corrections for net avoidance and flying height, it appeared important to determine the optimum radius for the incomplete ring of nets for a reliable efficiency estimate.     

The tsetse fly Glossina fuscipes fuscipes (Diptera: Glossina) harbours a surprising diversity of bacteria other than symbionts

Three different bacterial species are regularly described from tsetse flies. However, no broad screens have been performed to investigate the existence of other bacteria in this medically and agriculturally important vector insect. Utilising both culture dependent and independent methods we show that Kenyan populations of Glossina fuscipes fuscipes harbour a surprising diversity of bacteria. Bacteria were isolated from 72% of flies with 23 different bacterial species identified. The Firmicutes phylum dominated with 16 species of which seven belong to the genus Bacillus. The tsetse fly primary symbiont, Wigglesworthia glossinidia, was identified by the culture independent pathway. However, neither the secondary symbiont Sodalis nor Wolbachia was detected with either of the methods used. Two other bacterial species were identified with the DNA based method, Bacillus subtilis and Serratia marcescens. Further studies are needed to determine how tsetse flies, which only ever feed on vertebrate blood, pick up bacteria and to investigate the possible impact of these bacteria on Glossina longevity and vector competence.     

Metarhizium anisopliae infection reduces Trypanosoma congolense reproduction in Glossina fuscipes fuscipes and its ability to acquire or transmit the parasite

Background

Tsetse fly-borne trypanosomiasis remains a significant problem in Africa despite years of interventions and research. The need for new strategies to control and possibly eliminate trypanosomiasis cannot be over-emphasized. Entomopathogenic fungi (EPF) infect their hosts through the cuticle and proliferate within the body of the host causing death in about 3–14 days depending on the concentration. During the infection process, EPF can reduce blood feeding abilities in hematophagous arthropods such as mosquitoes, tsetse flies and ticks, which may subsequently impact the development and transmission of parasites. Here, we report on the effects of infection of tsetse fly (Glossina fuscipes fuscipes) by the EPF, Metarhizium anisopliae ICIPE 30 wild-type strain (WT) and green fluorescent protein-transformed strain (GZP-1) on the ability of the flies to harbor and transmit the parasite, Trypanosoma congolense.

Results

Teneral flies were fed T. congolense-infected blood for 2 h and then infected using velvet carpet fabric impregnated with conidia covered inside a cylindrical plastic tube for 12 h. Control flies were fed with T. congolense-infected blood but not exposed to the fungal treatment via the carpet fabric inside a cylindrical plastic tube. Insects were dissected at 2, 3, 5 and 7 days post-fungal exposure and the density of parasites quantified. Parasite load decreased from 8.7 × 10⁷ at day 2 to between 8.3 × 10⁴ and 1.3 × 10⁵ T. congolense ml^− 1 at day 3 post-fungal exposure in fungus-treated (WT and GZP-1) fly groups. When T. congolense-infected flies were exposed to either fungal strain, they did not transmit the parasite to mice whereas control treatment flies remained capable of parasite transmission. Furthermore, M. anisopliae-inoculated flies which fed on T. congolense-infected mice were not able to acquire the parasites at 4 days post-fungal exposure while parasite acquisition was observed in the control treatment during the same period.

Conclusions

Infection of the vector G. f. fuscipes by the entomopathogenic fungus M. anisopliae negatively affected the multiplication of the parasite T. congolense in the fly and reduced the vectorial capacity to acquire or transmit the parasite.

     

Polymorphic microsatellite markers for the tsetse fly Glossina fuscipes fuscipes (Diptera: Glossinidae), a vector of human African trypanosomiasis

Our understanding of Glossina fuscipes fuscipes, a major vector of sleeping sickness, has been severely constrained by a lack of genetic markers for mapping and population genetic studies. Here we present 10 newly developed microsatellite loci for this tsetse species. Heterozygosity levels in Moyo, an Ugandan population, averaged 0.57, with only two loci showing very low heterozygosity. Five loci carried more than six alleles. Together with five recently published microsatellite loci, this brings the number of available microsatellite loci for this species to 15. Their availability will greatly facilitate future studies on the genetics of this important human disease vector.