Prevalence of Trypanosoma and Sodalis in Wild Populations of Tsetse Flies: Impact on Sit Programmes for Tsetse Eradication

Tsetse flies, the vectors of African Trypanosoma, have a highly regulated and defined microbial fauna composed of three bacterial symbionts that may have a role to play in the establishment of Trypanosoma infections in the flies and hence, may influence the vectorial competence of the released sterile males. Sodalis bacteria seem to interact with Trypanosoma infection in tsetse flies. Field-caught tsetse flies of ten different taxa and from 15 countries were screened using PCR to detect the presence of Sodalis and Trypanosoma species and their interaction. The results indicate that the prevalence of Sodalis and Trypanosoma varied with country and tsetse species. Trypanosome prevalence was higher in east, central and southern African countries than in west African countries. Tsetse fly infection rates with Trypanosoma vivax and Trypanozoon spp were higher in west African countries, whereas tsetse infection with Trypanosoma congolense and T. simiae, T. simiae (tsavo) and T. godfreyi infection prevalence were higher in east, central and south African countries. Sodalis prevalence was high in Glossina morsitans morsitans and G. pallidipes but absent in Glossina tachinoides. Double and triple infections with Trypanosoma taxa and coinfection of Sodalis and Trypanosoma were rarely observed but it occurs in some taxa and locations. A significant Chi square value (< 0.05) seems to suggest that Sodalis and Trypanosoma infection correlate in Glossina palpalis gambiensis, Glossina pallidipes and Glossina medicorum. Trypanosoma infection significantly increased the density of Sodalis in wild G. m. morsitans and G. pallidipes flies however no significant impact of Sodalis infection on trypanosome density.

Interactions Between Glossina Pallidipes Salivary Gland Hypertrophy Virus and Tsetse Endosymbionts in Wild Tsetse Populations

BackgroundTherefore, tsetse control is considered an effective and sustainable tactic for the control of cyclically transmitted trypanosomosis in the absence of effective vaccines and inexpensive, effective drugs. The sterile insect technique (SIT) is currently used to eliminate tsetse fly populations in an area-wide integrated pest management (AW-IPM) context in Senegal. For SIT, tsetse mass-rearing is a major milestone that associated microbes can influence. Tsetse flies can be infected with micro-organisms, including the primary and obligate Wigglesworthia glossinidia, the commensal Sodalis glossinidius, and Wolbachia pipientis. In addition, tsetse populations often carry a pathogenic DNA virus, the Glossina pallidipes Salivary Gland Hypertrophy Virus (GpSGHV) that hinders tsetse fertility and fecundity. Interactions between symbionts and pathogens might affect the performance of the insect host. MethodsIn the present study, we assessed the possible interaction of GpSGHV and tsetse endosymbionts under field conditions to decipher the bidirectional interactions in different Glossina species. We determined the co-infection pattern of GpSGHV and Wolbachia in natural tsetse populations. We further analyzed the interaction of both Wolbachia and GpSGHV infection with Sodalis and Wigglesworthia density using qPCR. ResultsThe results indicated that the co-infection of GpSGHV and Wolbachia was most prevalent in Glossina austeni and Glossina morsitans morsitans, with an explicit significant negative correlation between GpSGHV and Wigglesworthia infection. GpSGHV infection levels of more than 104 were not observed when Wolbachia infection was present at high density (>108.5), suggesting a potential protective role of Wolbachia against GpSGHV. ConclusionThe result indicates that Wolbachia infection might protect tsetse fly against GpSGHV and the interactions between the tsetse host and its associated microbes are dynamic, likely species-specific and significant differences may exist between laboratory and field conditions.

Characterization and Tissue Tropism of Newly Identified Iflavirus and Negeviruses in Glossina morsitans morsitans Tsetse Flies

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host’s brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.

A gene expression panel for estimating age in males and females of the sleeping sickness vector Glossina morsitans

Many vector-borne diseases are controlled by methods that kill the insect vectors responsible for disease transmission. Recording the age structure of vector populations provides information on mortality rates and vectorial capacity, and should form part of the detailed monitoring that occurs in the wake of control programmes, yet tools for obtaining estimates of individual age remain limited. We investigate the potential of using markers of gene expression to predict age in tsetse flies, which are the vectors of deadly and economically damaging African trypanosomiases. We use RNAseq to identify candidate expression markers, and test these markers using qPCR in laboratory-reared Glossina morsitans morsitans of known age. Measuring the expression of six genes was sufficient to obtain a prediction of age with root mean squared error of less than 8 days, while just two genes were sufficient to classify flies into age categories of ≤15 and >15 days old. Further testing of these markers in field-caught samples and in other species will determine the accuracy of these markers in the field.

Effect of wing length on the prevalence of trypanosomes in Glossina morsitans morsitans in eastern Zambia

Background Tsetse flies (Diptera: Glossinidae) transmit trypanosomiasis (sleeping sickness in humans and nagana in livestock). Several studies have indicated that age, sex, site of capture, starvation and microbiome symbionts, among others, are important factors that influence trypanosome infection in tsetse flies. However, reasons for a higher infection rate in females than in males still largely remain unknown. Considering that tsetse species and sexes of larger body size are the most mobile and the most available to stationary baits, it was hypothesized in this study that the higher trypanosome prevalence in female than in male tsetse flies was a consequence of females being larger than males. Methods Black screen fly rounds and Epsilon traps were used to collect tsetse flies in eastern Zambia. Measurement of wing vein length and examination for presence of trypanosomes in the flies were carried out by microscopy. Principal component method was carried out to assess the potential of wing vein length as a predictor variable. The multilevel binary logistic regression method was applied on whole data, one-method data and one-sex data sets to evaluate the hypothesis. Results Data derived from a total of 2195 Glossina morsitans morsitans were evaluated (1491 males and 704 females). The wing length variable contributed the highest variance percentage (39.2%) to the first principal component. The variable showed significant influence on prevalence of trypanosomes when the analysis was applied on the whole data set, with the log odds for the prevalence of trypanosomes significantly increasing by 0.1 (P = 0.032), per unit increase in wing length. Females had higher trypanosome prevalence rates than males, though not always significant. Furthermore, moving from females to males, wing length significantly reduced by 0.2 (P < 0.0001). Conclusions We conclude that wing length is an important predictor variable for trypanosome prevalence in Glossina morsitans morsitans and could partially explain the higher prevalence of trypanosomes in females than in males. However, reasonably representative population data are required for analysis—a serious challenge with the current tsetse sampling methods. Thus, analysis combining data from mobile and stationary methods that include both sexes' data could be useful to verify this hypothesis. Graphical abstract

A novel vehicle-mounted sticky trap; an effective sampling tool for savannah tsetse flies Glossina morsitans morsitans Westwood and Glossina morsitans centralis Machado

Background Black screen fly round (BFR) is a mobile sampling method for Glossina morsitans. This technique relies on the ability of operator(s) to capture flies landing on the screen with hand nets. In this study, we aimed to evaluate a vehicle-mounted sticky panel trap (VST) that is independent of the operator’s ability to capture flies against BFR, for effective and rapid sampling of G. m. morsitans Westwood and G. m. centralis Machado. We also determined the influence of the VST colour (all-blue, all-black or 1:1 blue-black), orientation and presence of odour attractants on tsetse catch. Methodology/Principal findings Using randomised block design experiments conducted in Zambia, we compared and modelled the number of tsetse flies caught in the treatment arms using negative binomial regression. There were no significant differences in the catch indices of the three colour designs and for in-line or transversely oriented panels for both subspecies (P > 0.05). When baited with butanone and 1-octen-3-ol, VST caught 1.38 (1.11–1.72; P < 0.01) times more G. m. centralis flies than the un-baited trap. Attractants did not significantly increase the VST catch index for G. m. morsitans (P > 0.05). Overall, the VST caught 2.42 (1.91–3.10; P < 0.001) and 2.60 (1.50–3.21; P < 0.001) times more G. m. centralis and G. m. morsitans respectively, than the BFR. The VST and BFR took 10 and 35 min respectively to cover a 1 km transect. Conclusion/Significance The VST is several times more effective for sampling G. m. morsitans and G. m. centralis than the BFR and we recommend its use as an alternative sampling tool.

Effect of Wing Length on the Prevalence of Trypanosomes in Glossina Morsitans Morsitans in Eastern Zambia

BackgroundTsetse flies (Diptera: Glossinidae) transmit trypanosomiasis (sleeping sickness in humans and nagana in livestock). Several studies have indicated that the age, sex, site of capture, starvation and microbiome symbionts, among others, are important factors influencing trypanosome infection in tsetse flies. However, reasons for a higher infection rate in females than in males still largely remain unknown. As species and sexes of tsetse flies of larger body size are the most mobile and the most available to stationary baits, it was hypothesized in this study that, the higher trypanosome prevalence in female than in male tsetse flies was because females were larger than males.Materials and methodsBlack screen fly rounds and epsilon traps were used to collect tsetse flies in eastern Zambia. Wing vein length was measured and the presence of trypanosomes was examined by microscopy. Principal component method was carried out to determine which variables had potential to be used as predictor variables. The multilevel binary logistic regression method was applied on whole data, one-method data and one-sex data sets to evaluate the hypothesis. ResultsA total of 2,195 Glossina m. morsitans data were evaluated (1,491 and 704 males and females, respectively). The wing length variable contributed the highest variance percentage (39.15%) to the first principal component. The wing length variable showed significant influence on prevalence of trypanosomes when the whole data set was analyzed. The log odds for the prevalence of trypanosomes significantly increased by 0.123 (p = 0.032), per unit increase in wing length. Moreover, moving from females to males, wing length significantly reduced by 0.150 (p < 0.0001) and females had higher trypanosome prevalence rates than males, though not always significant. ConclusionWing length is an important determinant factor for trypanosome prevalence in G. m. morsitans and could partially explain the higher prevalence of trypanosomes in females than in males. To verify this status, however, it’s clear that, reasonably representative population data is required for analysis – a serious challenge with the current tsetse sampling methods. Nonetheless, analysis of combined data from mobile and stationary methods that includes both sexes data, could verify the status.

An atlas of tsetse and animal African trypanosomiasis in Zimbabwe

Background In the 1980s and 1990s, great strides were taken towards the elimination of tsetse and animal African trypanosomiasis (AAT) in Zimbabwe. However, advances in recent years have been limited. Previously freed areas have been at risk of reinvasion, and the disease in tsetse-infested areas remains a constraint to food security. As part of ongoing control activities, monitoring of tsetse and AAT is performed regularly in the main areas at risk. However, a centralized digital archive is missing. To fill this gap, a spatially explicit, national-level database of tsetse and AAT (i.e. atlas) was established through systematic data collation, harmonization and geo-referencing for the period 2000–2019. Methods The atlas covers an area of approximately 70,000 km2, located mostly in the at-risk areas in the north of the country. In the tsetse component, a total of 33,872 entomological records were assembled for 4894 distinct trap locations. For the AAT component, 82,051 samples (mainly dry blood smears from clinically suspicious animals) were collected at 280 diptanks and examined for trypanosomal infection by microscopy. Results Glossina pallidipes (82.7% of the total catches) and Glossina morsitans morsitans (17.3%) were the two tsetse species recorded in the north and northwest parts of the country. No fly was captured in the northeast. The distribution of AAT follows broadly that of tsetse, although sporadic AAT cases were also reported from the northeast, apparently because of transboundary animal movement. Three trypanosome species were reported, namely Trypanosoma brucei (61.7% of recorded infections), Trypanosoma congolense (28.1%) and Trypanosoma vivax (10.2%). The respective prevalences, as estimated in sentinel herds by random sampling, were 2.22, 0.43 and 0.30%, respectively. Discussion The patterns of tsetse and AAT distributions in Zimbabwe are shaped by a combination of bioclimatic factors, historical events such as the rinderpest epizootic at the turn of the twentieth century and extensive and sustained tsetse control that is aimed at progressively eliminating tsetse and trypanosomiasis from the entire country. The comprehensive dataset assembled in the atlas will improve the spatial targeting of surveillance and control activities. It will also represent a valuable tool for research, by enabling large-scale geo-spatial analyses.

Effects of Human Settlements and Spatial Distribution of Wing Vein Length, Wing Fray Categories and Hunger Stages in Glossina morsitans morsitans (Diptera: Glossinidae) and Glossina pallidipes (Diptera: Glossinidae) in Areas Devoid of Cattle in North-Eastern Zambia

The effect of human-associated habitat degradation on tsetse populations is well established. However, more insights are needed into how gradual human encroachment into tsetse fly belts affect tsetse populations. This study investigated how wing vein length, wing fray categories, and hunger stages, taken as indicators of body size, age, and levels of access to hosts, respectively, in Glossina morsitans morsitans Westwood (Diptera: Glossinidae) and Glossina pallidipes Austen (Diptera: Glossinidae), varied along a transect from the edge into inner parts of the tsetse belt, in sites that had human settlement either concentrated at the edge of belt or evenly distributed along transect line, in north-eastern Zambia. Black-screen fly round and Epsilon traps were used in a cross-sectional survey on tsetse flies at three sites, following a transect line marked by a road running from the edge into the inner parts of the tsetse belt, per site. Two sites had human settlement concentrated at or close to the edge of the tsetse belt, whereas the third had human settlement evenly distributed along the transect line. Where settlements were concentrated at the edge of tsetse belt, increase in distance from the settlements was associated with increase in wing vein length and a reduction in the proportion of older, and hungry, tsetse flies. Increase in distance from human settlements was associated with improved tsetse well-being, likely due to increase in habitat quality due to decrease in effects of human activities.